CA3160986A1 - Lentiviral vectors in hematopoietic stem cells to treat wiskott-aldrich syndrome (was) - Google Patents

Abstract

In certain embodiments a lentiviral vector for the treatment of Wiskott-Aldrich Syndrome (WAS) is provided. In certain embodiments the vector comprises an expression cassette comprising a nucleic acid construct comprising an effective fragment of the endogenous promoter of the WAS gene where said promoter has maximum length of 600 bp and contains the sequence of HS1pro, and a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp) operably linked to the effective fragment of the endogenous promoter of the WAS gene.

Description

LENTIVIRAL VECTORS IN HEMATOPOIETIC STEM CELLS TO
TREAT WISKOTT-ALDRICH SYNDROME (WAS) CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of USSN
62/933,875, filed on November 11, 2019, which is incorporated herein by reference in its entirety for all purposes.
STATEMENT OF GOVERNMENTAL SUPPORT
[ Not Applicable ]
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A
TEXT FILE

[0002] A Sequence Listing is provided herewith as a text file, "UCLA-P220P_ST25.txt" created on November 9, 2020 and having a size of 82.1 kb. The contents of the text file are incorporated by reference herein in their entirety.
BACKGROUND

[0003] Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immune deficiency caused by mutations in the Wiskott-Aldrich Syndrome (WAS) gene. Patients with WAS have severe defects in both adaptive and innate immunity and are highly susceptible to life-threatening viral and bacterial infections. Patients also suffer from extremely severe eczema, microthrombocytopenia and have a high risk of developing autoimmunity and cancer,

[0004] WAS occurs most often in males due to its X-linked recessive pattern of inheritance, affecting between 1 and 10 males per million. The first signs are usually petechiae and bruising, resulting from a low platelet count (i.e, thrombocytopenia).
Spontaneous nose bleeds and bloody diarrhea are also common, and eczema typically develops within the first month of life. Recurrent bacterial infections typically develop by three months. The majority of children with WAS develop at least one autoimmune disorder, and cancers (mainly lymphoma and leukemia) develop in up to a third of patients.
Immunoglobulin M (IgM) levels are typically reduced, IgA and IgE are typically elevated, and IgG levels can be normal, reduced, or elevated. In addition to thrombocytopenia, WAS
patients have abnormally small platelets (i.e. microthrombocytes) and ¨30%
also have elevated eosinophil counts (i.e., eosinophilia).

[0005] Treatment for WAS typically involves prophylactic antibiotic and antiviral therapy to manage infections. Platelet transfusions and splenectomy are used to treat thrombocytopenia (low platelet counts). Additionally, blood transfusions may be required to treat anemia resulting from excessive bleeding and a protective helmet may be used to prevent brain hemorrhages which could result from head injury.
Immunosuppressive treatment is utilized for autoimmune manifestations.

[0006] Because patients with WAS have abnormal T- and B-lymphocyte function, they are typically not administered live virus vaccines since there is a possibility that a vaccine strain of the virus may cause disease. Complications of chicken pox infection occur occasionally and may be prevented by early treatment following exposure with antiviral drugs, high dose immunoglobulin replacement therapy or Varicella zoster Immune Globulin (VZIG). Other "non-live" vaccinations can be given safely to patients with WAS
but may not generate protective levels of antibody.

[0007] A potential curative therapy is an allogeneic hematopoietic stem cell transplantation from a HLA matched donor. However, this is not a viable option for many patients due to the unavailability of a suitable matched donor.

[0008] An alternative curative therapy is an autologous hematopoietic stem cell (HSC) transplantation with ex vivo gene therapy. In this approach, the patient acts as their own donor, eliminating the risk of immunological complications. Successful implementation of this approach relies on the development of a lentiviral vector or CRISPR
based therapy to introduce a functional copy of the gene of interest or to correct the pathogenic mutation in the patient's HSCs.

[0009] Previous viral-based therapies utilized the CMMP-WAS y-retroviral vector.
This therapy restored function and platelet counts in all patients. However, 7/9 patients developed acute leukemia due to insertional oncogenesis (see, e.g., Braun (2014) Sci. Transl.
Med. 6(227): 227ra33).

[0010] Another therapy uses a safer SIN lentiviral vector driven by a 1.6kb promoter fragment of the endogenous WAS gene. Using this vector, about 6/7 patients showed clinical improvement post gene therapy. T, B, and NK cells were functional and normal immune cell counts were restored. Additionally, there was a decrease in severity and frequency of infections and severe eczema was resolved. However, platelet counts and mean platelet size remain under normal values. The patients remained microthrombocytopenic and retained a risk of severe bleeding episodes.

SUMMARY

[0011] Described herein is the development of novel lentiviral vector(s) (LVs) for the treatment of Wiskott-Aldrich Syndrome (WAS). The vectors described herein show better (higher) expression than the current lentiviral vector in megakaryocytes and are believed to be able to restore platelet counts to normal levels in WAS patients.
Additionally, the vectors described herein are believed to maintain levels of expression similar to the previous SIN LV
expressing a WAS gene in all other hematopoietic cell lineages and thus is believed to be able to restore T, B and NK cell counts and function).

[0012] Accordingly, various embodiments contemplated herein may include, but need not be limited to, one or more of the following:

[0013] Embodiment 1: A recombinant lentiviral vector (LV) for the treatment of Wiskott-Aldrich Syndrome (WAS), said vector comprising:

[0014] an expression cassette comprising:

[0015] a nucleic acid encoding an effective fragment of the endogenous promoter of the WAS gene where said promoter has maximum length of 600 bp and contains the sequence of HS 1pro (SEQ ID NO:1); and

[0016] a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp) operably linked to said effective fragment of the endogenous promoter of the WAS gene.

[0017] Embodiment 2: The vector of embodiment 1, wherein the sequence of said effective fragment of the endogenous promoter of the WAS gene consists of the sequence of HS 1pro (SEQ ID NO:1).

[0018] Embodiment 3: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises a slim enhancer element 2 (SEQ ID
NO:2 = SEQ
ID NOs:3-8) or an effective fragment thereof.

[0019] Embodiment 4: The vector of embodiment 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).

[0020] Embodiment 5: The vector of embodiment 4, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID
NO:8).

[0021] Embodiment 6: The vector of embodiment 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID
NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).

[0022] Embodiment 7: The vector of embodiment 6, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).

[0023] Embodiment 8: The vector of embodiment 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the 1 st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID
NO:3).

[0024] Embodiment 9: The vector according to any one of embodiments 3 and 8, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the second half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4).

[0025] Embodiment 10: The vector according to any one of embodiments 3 and 8-9, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5).

[0026] Embodiment 11: The vector according to any one of embodiments 3 and 8-10, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6).

[0027] Embodiment 12: The vector according to any one of embodiments 3 and 8-11, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7).

[0028] Embodiment 13: The vector according to any one of embodiments 3 and 8-12, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO:8).

[0029] Embodiment 14: The vector according to any one of embodiments 1-13, wherein said expression cassette comprises enhancer element H53 (SEQ ID NO:9) or an effective fragment thereof.

[0030] Embodiment 15: The vector of embodiment 14, wherein said expression cassette comprises an effective fragment of enhancer element H53 wherein said fragment comprises or consists of H53 core sequence (SEQ ID NO: 10).

[0031] Embodiment 16: The vector of embodiment 15, wherein said expression cassette comprises an effective fragment of enhancer element H53 wherein said fragment consists of H53 core sequence (SEQ ID NO: 10).

[0032] Embodiment 17: The vector according to any one of embodiments 1-16, wherein said expression cassette comprises enhancer element E9 (SEQ ID NO:11) or an effective fragment thereof.

[0033] Embodiment 18: The vector of embodiment 17, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment comprises or consists of enhancer element E9 core sequence (SEQ ID NO:12).

[0034] Embodiment 19: The vector of embodiment 18, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment consists of enhancer element E9 core sequence (SEQ ID NO:12).

[0035] Embodiment 20: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises: a slim enhancer element 2 (SEQ ID
NO:2 = SEQ
ID NOs:3-8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS 1pro (SEQ ID NO:1).

[0036] Embodiment 21: The vector of embodiment 20, wherein said vector comprises the features shown in Figure 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp)..

[0037] Embodiment 22: The vector of embodiment 20, wherein said vector comprises the sequence show in SEQ ID NO:15 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp)..

[0038] Embodiment 23: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises:

[0039] enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO:12);

[0040] enhancer element H53 sequence comprising or consisting of H53 core sequence (SEQ ID NO: 10);

[0041] a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8); and

[0042] a fragment of the endogenous promoter of the WAS gene comprising or consisting of the sequence of HS 1pro (SEQ ID NO:1).

[0043] Embodiment 24: The vector of embodiment 23, wherein said vector comprises the features shown in Figure 19 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0044] Embodiment 25: The vector of embodiment 23, wherein said vector comprises the sequence show in SEQ ID NO:16 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0045] Embodiment 26: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises:

[0046] enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO:12);

[0047] enhancer element H53 sequence comprising or consisting of H53 core sequence (SEQ ID NO: 10);

[0048] enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID
NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and

[0049] a fragment of the endogenous promoter of the WAS gene comprising or consisting of the sequence of HS 1pro (SEQ ID NO:1).

[0050] Embodiment 27: The vector of embodiment 26, wherein said vector comprises the features shown in Figure 20 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0051] Embodiment 28: The vector of embodiment 26, wherein said vector comprises the sequence show in SEQ ID NO:17 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0052] Embodiment 29: The vector according to any one of embodiments 1-2, wherein said expression cassette comprises:

[0053] enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO:12);

[0054] enhancer element H53 sequence comprising or consisting of H53 core sequence (SEQ ID NO: 10); a first half of enhancer element 2 core sub-element 1 (SEQ ID
NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and

[0055] a fragment of the endogenous promoter of the WAS gene comprising or consisting of the sequence of HS 1pro (SEQ ID NO:1).

[0056] Embodiment 30: The vector of embodiment 29, wherein said vector comprises the features shown in Figure 21 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0057] Embodiment 31: The vector of embodiment 29, wherein said vector comprises the sequence show in SEQ ID NO:18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0058] Embodiment 32: The vector according to any one of embodiments 1-31, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS
cDNA or a codon-optimized WAS gene.

[0059] Embodiment 33: The vector of embodiment 32, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS cDNA (SEQ ID NO:13).

[0060] Embodiment 34: The vector of embodiment 32, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a codon optimized WAS.

[0061] Embodiment 35: The vector of embodiment 34, wherein the sequence of said nucleic acid that encodes WASP is a codon optimized WAS selected from the group consisting of jCAT codon optimized WAS, GeneArt optimized WAS, and IDT
optimized WAS.

[0062] Embodiment 36: The vector according to any one of embodiments 1-35, wherein said vector comprises a iv region vector genome packaging signal.

[0063] Embodiment 37: The vector according to any one of embodiments 1-36, wherein said vector comprise a 5 LTR comprising a CMV enhancer/promoter.

[0064] Embodiment 38: The vector according to any one of embodiments 1-37, wherein said vector comprises a Rev Responsive Element (RRE).

[0065] Embodiment 39: The vector according to any one of embodiments 1-38, wherein said vector comprises a central polypurine tract.

[0066] Embodiment 40: The vector according to any one of embodiments 1-39, wherein said vector comprises a post-translational regulatory element.

[0067] Embodiment 41: The vector of embodiment 40, wherein the posttranscriptional regulatory element is modified Woodchuck Post-transcriptional Regulatory Element (WPRE).

[0068] Embodiment 42: The vector according to any one of embodiments 1-41, wherein said vector is incapable of reconstituting a wild-type lentivirus through recombination.

[0069] Embodiment 43: The vector according to any one of embodiments 1-42, wherein said vector shows high expression in megakaryocytes.

[0070] Embodiment 44: The vector according to any one of embodiments 1-43, wherein said vector restores T, B and NK cell counts and function when administered to a mammal having WAS.

[0071] Embodiment 45: A host cell transduced with a vector according to any one of embodiments 1-44.

[0072] Embodiment 46: The host cell of embodiment 45, wherein the cell is a stem cell.

[0073] Embodiment 47: The host cell of embodiment 46, wherein said cell is a stem cell derived from bone marrow, and/or from umbilical cord blood, and/or from peripheral blood.

[0074] Embodiment 48: The host cell of embodiment 45, wherein the cell is a human hematopoietic progenitor cell.

[0075] Embodiment 49: The host cell of embodiment 48, wherein the human hematopoietic progenitor cell is a CD34+ cell.

[0076] Embodiment 50: A method of treating Wiskott-Aldrich Syndrome (WAS), in a subject, said method comprising:

[0077] transducing a stem cell and/or progenitor cell from said subject with a vector according to any one of embodiments 1-44; and

[0078] transplanting said transduced cell or cells derived therefrom into said subject where said cells or derivatives therefrom express said WASp protein.

[0079] Embodiment 51: The method of embodiment 50, wherein the cell is a stem cell.

[0080] Embodiment 52: The host cell of embodiment 50, wherein said cell is a stem cell derived from bone marrow.

[0081] Embodiment 53: The method of embodiment 50, wherein the cell is a human hematopoietic stem and progenitor cell.

[0082] Embodiment 54: The method of embodiment 53, wherein the human hematopoietic progenitor cell is a CD34+ cell.

[0083] Embodiment 55: A recombinant nucleic acid comprising one or more of the following:

[0084] a nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HS 1pro (SEQ ID NO:1); and/or

[0085] a nucleic acid sequence comprising or consisting of a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof; a nucleic acid sequence comprising or consisting of a 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO: 3) ; and/or

[0086] a nucleic acid sequence comprising or consisting of a 2nd half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4); and/or

[0087] a nucleic acid sequence comprising or consisting of a Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5); and/or

[0088] a nucleic acid sequence comprising or consisting of a Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO: 6); and/or

[0089] a nucleic acid sequence comprising or consisting of a Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7); and/or

[0090] a nucleic acid sequence comprising or consisting of a Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8); and/or

[0091] a nucleic acid sequence comprising or consisting of enhancer element H53 (full) (SEQ ID NO:9) or an effective fragment thereof; and/or

[0092] a nucleic acid sequence comprising or consisting of Enhancer element H53 core (SEQ ID NO:10); and/or

[0093] a nucleic acid sequence comprising or consisting of Enhancer element E9 (full) (SEQ ID NO:11) or an effective fragment thereof; and/or

[0094] a nucleic acid sequence comprising or consisting of Enhancer element E9 core (SEQ ID NO:12).

[0095] Embodiment 56: The nucleic acid of embodiment 55, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HS 1pro (SEQ ID NO:1).

[0096] Embodiment 57: The nucleic acid of embodiment 55, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof.

[0097] Embodiment 58: The nucleic acid of embodiment 55, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO: 3).

[0098] Embodiment 59: The nucleic acid according to any one of embodiments 55, and 58, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 2nd half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4).

[0099] Embodiment 60: The nucleic acid according to any one of embodiments 55, and 58-59, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5).

[0100] Embodiment 61: The nucleic acid according to any one of embodiments 55, and 58-60, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6).

[0101] Embodiment 62: The nucleic acid according to any one of embodiments 55, and 58-61, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7).

[0102] Embodiment 63: The nucleic acid according to any one of embodiments 55, and 58-62, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8).

[0103] Embodiment 64: The nucleic acid according to any one of embodiments 55, and 58-63, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of enhancer element H53 (full) (SEQ ID NO:9) or an effective fragment thereof.

[0104] Embodiment 65: The nucleic acid according to any one of embodiments 55, and 58-64 , wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element HS3 core (SEQ ID NO:10).

[0105] Embodiment 66: The nucleic acid according to any one of embodiments 55, and 58-65, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 (full) (SEQ ID NO:11) or an effective fragment thereof.

[0106] Embodiment 67: The nucleic acid according to any one of embodiments 55, and 58-66, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 core (SEQ ID NO:12).

[0107] Embodiment 68: The nucleic acid according to any one of embodiments 67, wherein said nucleic acid comprises an expression cassette.

[0108] Embodiment 69: The nucleic acid of embodiment 68, wherein said expression cassette is effective to express WASp when transduced into a mammalian cell.

[0109] Embodiment 70: The nucleic acid of embodiment 55, wherein said nucleic acid comprises a vector according to any one of embodiments 1-44.
Definitions.

[0110] A "promoter" refers to a regulatory sequence in a nucleic acid required to initiate transcription of a gene (e.g., a gene operably coupled to the promoter).

[0111] An "enhancer" refers to a regulatory DNA sequence that, when bound by specific proteins called transcription factors, enhance the transcription of an associated gene.

[0112] An "effective fragment" when used with respect to a promoter (e.g., an effective fragment of a WAS promoter) refers to a fragment of the full-length promoter that is sufficient to initiate transcription of a gene operably linked to that promoter.

[0113] An "effective fragment" when used with respect to an enhancer (e.g., an effective fragment of a WAS enhancer) refers to a fragment of the full-length enhancer that is sufficient to provide regulate expression of an operably linked gene when bound by a transcription factor. In certain embodiments the regulation is comparable with respect to expression level and/or lineage offered by the full-length enhancer.

[0114] The term "operably linked" refers to a nucleic acid sequence placed into a functional relationship with another nucleic acid sequence. For example, a promoter is operably linked to a gene when that promoter is placed in a location that permits that promoter to initiate transcription of that gene. An enhancer is operably linked to a gene when that enhancer, when bound by an appropriate transcription factor, is able to regulate (e.g., to upregulate) expression of that gene.

[0115] "Recombinant" is used consistently with its usage in the art to refer to a nucleic acid sequence that comprises portions that do not naturally occur together as part of a single sequence or that have been rearranged relative to a naturally occurring sequence. A
recombinant nucleic acid is created by a process that involves the hand of man and/or is generated from a nucleic acid that was created by hand of man (e.g., by one or more cycles of replication, amplification, transcription, etc.). A recombinant virus is one that comprises a recombinant nucleic acid. A recombinant cell is one that comprises a recombinant nucleic acid.

[0116] As used herein, the term "recombinant lentiviral vector" or "recombinant LV) refers to an artificially created polynucleotide vector assembled from an LV
and a plurality of additional segments as a result of human intervention and manipulation.

[0117] By an effective amount" is meant the amount of a required agent or composition comprising the agent to ameliorate or eliminate symptoms of a disease relative to an untreated patient. The effective amount of composition(s) used to practice the methods described herein for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject.
Ultimately, the .. attending physician or veterinarian will decide the appropriate amount and dosage regimen.
Such amount is referred to as an "effective" amount.
BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1, panel A, shows illustrative LV constructs used to evaluate regulatory activity of various combinations of elements HS1, H52, H53, and H54. Panel B
shows a construct used to evaluate the combination of the E3 element and HS1.

[0119] Figure 2 shows expression levels in MEG-01 cells transduced with WAS
vectors.

[0120] Figure 3 shows expression levels of WAS vectors in Jurkat cells (T-cell line).

[0121] Figure 4 shows expression levels of WAS vectors in RAMOs cells (B-cell line).

[0122] Figure 5, panels A-B, illustrates the constructs used to identify the critical enhancer elements that regulate the WAS gene. Panel A) Ten new constructs each containing a putative enhancer element cloned upstream of the endogenous minimal WAS
promoter (HS 1pro). Panel B) The gamma-retroviral vector (CMMP-mCit) which was able to restore platelet counts to normal levels and used as a control.

[0123] Figure 6 shows expression levels of WAS vectors in MEG-01 (megakaryoblast cell line).

[0124] Figure 7 shows expression levels of WAS vectors in Jurkat cells (T-cell line).

[0125] Figure 8 shows expression levels of WAS vectors in RAMOs cells (B-cell line).

[0126] Figure 9 shows expression levels of WAS vectors in CB CD34+
differentiated megakaryocytes "pro-megakaryocytes".

[0127] Figure 10 shows expression levels of WAS vectors in CB CD34+
differentiated megakaryocytes "megakaryocytes".

[0128] Figure 11 shows expression levels of WAS vectors in CB CD34+
differentiated megakaryocytes "platelets".

[0129] Figure 12 WAS vectors in CB CD34+ differentiated megakaryocytes "pro-megakaryocytes".

[0130] Figure 13 shows expression levels of WAS vectors in CB CD34+
differentiated megakaryocytes "megakaryocytes".

[0131] Figure 14 shows expression levels of WAS vectors in CB CD34+
differentiated megakaryocytes "platelets".

[0132] Figure 15 shows expression levels of WAS vectors comprising each of 5 sub-elements of E2 in CB CD34+ differentiated megakaryocytes "megakaryocytes".

[0133] Figure 16 shows expression levels of WAS vectors comprising each of 5 E2 fragments in CB CD34+ differentiated megakaryocytes "megakaryocytes".

[0134] Figure 17 shows expression levels of WAS vectors comprising each of fragments in in CB CD34+ differentiated megakaryocytes "platelets".

[0135] Figure 18 shows a schematic of the E2(all slim) vector (E2(all slim)-HS1pro-mCit-WPRE).

[0136] Figure 19 shows a schematic of the E9(slim)-H53(slim)-E2(all slim)-HS 1pro-mCit-WPRE vector in which the E9(slim) and H53(slim) elements have been added.

[0137] Figure 20 shows a schematic of the E9(slim)-HS3(slim)-E2(1,4,5 slim)-HS 1pro-mCit-WPRE (Deleted Core Sub-Element 2 of Element 2 and Core Sub-Element 3 of Element 2). E9slimHS3slimE2145slimHS1pro

[0138] Figure 21 shows a schematic of the E9(slim)-HS3(slim)-E2(1' half of 1 and 5 slim)-HS 1pro-mCit-WPRE (Deleted 2nd half of Core Sub-Element 1 of Element 2, Core Sub-Element 2 of Element 2 and Core Sub-Element 3 of Element 2, and Core-Sub Element 4 of Element 2).

[0139] Figure 22 shows expression of the candidate vectors in CB CD34+

differentiated megakaryocytes.

[0140] Figure 23 shows expression of the candidate vectors in CB CD34+
differentiated platelets.

[0141] Figure 24 shows screening of codon optimizations in an immortalized WAS
patient B-cell line.

[0142] Figure 25 shows screening of codon optimizations in an immortalized WAS
patient T-cell line.

[0143] Figure 26 shows titer produced by codon optimized versions of WAS Vec.
DETAILED DESCRIPTION

[0144] In various embodiments, lentiviral vectors are provided for the treatment (or prophylaxis) of Wiskott-Aldrich Syndrome (WAS) are provided. In certain embodiments the vectors are optimized to reduce vector size, increase expression level and titer. Additionally, in various embodiments the vectors to recapitulate the expression pattern of the native WAS
gene, e.g., as described herein.

[0145] In particular, a bioinformatic analysis (using publicly available databases:
Project Encode, Ensemnbl, FANTOM, VISTA Enhancer Browser, GeneHancer) was utilized to elucidate the endogenous regulatory elements of the native WAS gene.
Thirteen putative endogenous enhancer elements were identified contained within a 1.1 million base pair window.

[0146] A lentiviral library was constructed in order to experimentally validate each of the putative regulatory elements. Each element was validated in MEG-01 cells (Megakaryocyte cell line), Jurkats (T-cell line), RAMOs (B-cell line) as well as in a cord blood CD34+ differentiated megakaryocytes for enhancer activity.

[0147] Further experiments were done to identify necessary (core) region(s) of the enhancers and the WAS endogenous promoter and the combinations of minimal promoter and reduced (slim) enhancer elements necessary to achieve suitable expression of the WAS
gene were identified. The validated enhancer elements will be used to design our lead candidate lentiviral vector for the treatment of WAS.

[0148] Accordingly, in certain embodiments a recombinant lentiviral vector (LV) for the treatment of Wiskott-Aldrich Syndrome (WAS) is provided where the vector comprises an expression cassette comprising a nucleic acid encoding an effective fragment of the endogenous promoter of the WAS gene where the promoter has maximum length of 600 bp and contains the sequence of HS 1pro (SEQ ID NO:1); and a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp) operably linked to the effective fragment of the endogenous promoter of the WAS gene. In certain embodiments the effective fragment of the endogenous promoter of the WAS gene consists of the sequence of HS 1pro (SEQ
ID NO:1).

[0149] In certain embodiments the expression cassette comprises a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof. In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment comprises or consists of enhancer element 2 core sub-element 1 (SEQ ID
NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8). In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ
ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8). In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment comprises or consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8). In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).

[0150] In certain embodiments the expression cassette comprises an effective fragment of enhancer element 2 where the fragment comprises or consists of the 1' half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:3), and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of the second half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4), and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO: 5); and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core Sub-Element 3 of Enhancer Element 2 (SEQ ID
NO :6); and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7); and/or an effective fragment of enhancer element 2 where the fragment comprises or consists of Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO:8).

[0151] In certain embodiments the expression cassette comprises enhancer element H53 (SEQ ID NO:9) or an effective fragment thereof. In certain embodiments the expression cassette comprises an effective fragment of enhancer element H53 where the fragment comprises or consists of H53 core sequence (SEQ ID NO: 10). In certain embodiments the expression cassette comprises an effective fragment of enhancer element H53 where the fragment consists of H53 core sequence (SEQ ID NO: 10).

[0152] In certain embodiments the expression cassette comprises enhancer element E9 (SEQ ID NO:11) or an effective fragment thereof. In certain embodiments the expression cassette comprises an effective fragment of enhancer element E9 where the fragment comprises or consists of enhancer element E9 core sequence (SEQ ID NO:12). In certain embodiments the expression cassette comprises an effective fragment of enhancer element E9 where the fragment consists of enhancer element E9 core sequence (SEQ ID
NO:12).

[0153] In certain embodiments the expression cassette comprises a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8), and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS 1pro (SEQ ID NO:1). In certain embodiments this vector comprises the features shown in Figure 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO:15 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0154] In certain embodiments the expression cassette comprises enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO:12), enhancer element H53 sequence comprising or consisting of H53 core sequence (SEQ ID NO:
10), a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS 1pro (SEQ
ID
NO:1). In certain embodiments this vector comprises the features shown in Figure 19 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO:16 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0155] In certain embodiments the expression cassette comprises an enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO:12), enhancer element H53 sequence comprising or consisting of H53 core sequence (SEQ ID NO:
10), enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID
NO:8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS 1pro (SEQ ID NO:1). In certain embodiments this vector comprises the features shown in Figure 20 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO:17 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0156] In certain embodiments the expression cassette comprises enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO:12), enhancer element H53 sequence comprising or consisting of H53 core sequence (SEQ ID NO:
10), a first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and a fragment of the endogenous promoter of the WAS
gene consisting of the sequence of HS 1pro (SEQ ID NO:1). In certain embodiments this vector comprises the features shown in Figure 21 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp). In certain embodiments this vector comprises the sequence show in SEQ ID NO:18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

[0157] It will be recognized that, for clinical use, the mCitrine reporter can be replaced with a nucleic acid encoding a WASp protein. In certain embodiments such a nucleic acid is a WAS cDNA or a codon-optimized WAS gene (e.g., a jCAT
optimized WAS).
In certain embodiments the sequence of the nucleic acid that encodes WASP is a codon optimized WAS selected from the group consisting of jCAT codon optimized WAS, GeneArt optimized WAS, and IDT optimized WAS.

[0158] It will also be recognized that the expression cassettes described herein with respect to lentiviral vectors need not be limited to this use, and can be incorporated in essentially any other construct (e.g., a CRISPR construct) where expression of a WASp is desired. Thus, in certain embodiments, nucleic acid constructs comprising any of the expression cassette components described herein are contemplated.

Table 1. Nucleic acid sequences of various components of the lentiviral vectors for treatment of WAS as described herein.
Element Nucleic Acid Sequence SEQ ID NO
TCAGCCTCAGGCTACCTAGGTGCTTTAGAAAGGAGGCCACCC
HS 1pro (what AGGCCCATGACTACTCCTTGCCACAGGGAGCCCTGCACACAG
we define as the ATGTGCTAAGCTCTCGCTGCCAGCCAGAGGGAGGAGGGTCTG
minimal AGCCAGTCAGAAGGAGATGGGCCCCAGAGAGTAAGAAAGGG
endogenous GGAGGAGGACCCAAGCTGATCCAAAAGGTGGGTCTAAGCAG
promoter of the TCAAGTGGAGGAGGGTTCCAATCTGATGGCGGAGGGCCCAA
WAS gene) GCTCAGCCTAACGAGGAGGCCAGGCCCACCAAGGGGCCCCT
(SEQ ID NO: 1) GGAGGACTTGTTTCCCTTGTCCCTTGTGGTTTTTTGCATTTCCT
GTTCCCTTGCTGCTCATTGCGGAAGTTCCTCTTCTTACCCTGC
ACCCAGAGCCTCGCCAGAGAAGACAAGGGCAGAAAGCACC
AGGCAAAGGGCAGTTCACACCAAAGTCCACCCCCAGCTCAGG
GCCCGATACAGTAAAGGAAATCAGGAAAAGTTTACTGAGCA
AATGGATAGGCTGGGTAAGCCAGGGAGCTCTTCTCCCTGTGA
AATTAAAGTGGGCCTGAAGATCATGCAAGTCTGATTTCCACT
GGTTTTGGGAATTTCCCTTTGTTTCCCCAGAGGACCACTGCTC
TGATGGGATCTCCCTGGGGCATGGGGCTAGGGTCAGGTCACA
CAGGAACACTCCTGAGTTCTTCCCTGTCAGTCTACCTCTCTGA
AGAACTGTCAGAAGAACACCTTCCCTGCCTTCCCCTTCCTACC
CTGGAGTGTTCTGTGAGAAAGGCTACTCTGAGATAGAAAATA
GGGCCCATGTTTGTCTCCAGTCCTCTTGGAGGTGAAGAGACC
TGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGG
GGGTAGCCCCTTCACCAATGTAAACAAGGATGTGGGTTCTGC
GGCCACACTCTCCCCCGCCCTCCCCAGCGGCATTTCCAGCAA
GTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCTGCTGC
CTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTA
CTTGGGGAGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGA
Enhancer GGCAGTGCTGTTGGCAGCTGCAGTAAACAGGAACAAAGATG
Element 2 (Full) AGAATGAAGGGGTTAAATAGGGGTTGGCTGGCCAGGCTCAG
AGAGATAGGCTGAGGTGCTGGAGAAAGCCCATCTCCTATGGA
AAGGTCAACGGTCATTTCCAGGCCTGGGAGTTCTATCTTCAA
GAGCTAGATGCCAGATGCAGGAAAATGGGGAAAGAGAGGAG
ACTGTGGAACAGGGAAGGGATCTGGGGGAAAACCCCCTGAG
GGTCCCATTTCTCTGCCACTGACCCCTTTGCCTGGCAGAAAGG
CTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAA
CCTCTCTAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAG
TAACTCCTCTTCCATCTGCTGGGTGTTGTCAAAACTACACAGG
AGGTTTGCCAAGCACCTAAGGCACAGTAGGTGGTCAAGAAAC
AGGAACACAATTTGTCATGACGACAATGATTCCTTCATATGC
TAGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTA
TGAAGCCCTTCCTCATTTCTGTTGCAATTCCTCAGCCAGAGGA
AAGCCCCAAATGCAGACCTAGGATGCATGTCCTCTCCTGGCT
CTCTACCTTTAGCGAAACCAAAGGTCCACAAGTCTACCTCTA
GGCCGTTAGTTCTAACAAAACTTCAGCAGTTAAATTAATATC
GAATTAACAATACAGGCTTCAAACTCTGTCTCAAAAAAAAAA

AAAAGAATTCGTTTTTCTGACTTTCCTAGCTCCACCTCTGTCC
TCCAGAAAGCCCTCCTCTTCCCAGAAGGCTGGCACACAGGGC
TGGGGGCCAGGCTTGACTTCCCTGTGGAGGGGTCTGAGGTTG
GGGGGATGACCTGCAGAGGTAGGGGAATTCAGAGAACTTGTT
TTGATAGAAACTGAAACTCCTCCTCTTGCTGACGCTGCACTTG
TGGGCAATCTGCACCTGTTCTCCCCTCCCAGAGGCCAGCTGG
AAGGGGGTCCCCCACCATTCTACCTCTGTTCCCATCTCAAACA
GGGCTCAGAAATCCTCAGTACAATACTACAATTGGTCCCACA
CCAAAGACTTTTCACACTCCAACTCATTCTATCCTTACCAAAA
CACTCTTACTTTACCCAGGAGACACAGAAATGTGGCAAAGCT
TGCCTGGGATCACAGATTTAGGGGGAGTGAAGGGGAGGCCA
GGCTCTCAAAGGGTAACTGCTGGCCCCAGCTGATACCCAGAT
TTATGTTAAGAAGCAAGACATGAGAGCCTGGATTATCTTCCT
ATAGGGTCCTACCATTTGCTTTAGAGACATCTGAGAACTGCCT
ACTCCCCAGGTAGCTGCCTGCCTCCCAGAAGAGCCACAGCAA
GTTCCGCTAAGGGCAAAAAAGGAAGCTAGGTGTCTACAGGG
AACCTAAAAACAAACCACACTAACGTGTGTACACACAGCTGC
AGAGGGAGACAGGGACCTGGTCTGCTCCCCACTACCCTCCTG
GTCCAGTGGGAACCCTGGCCCCACTGAGAACCAATCTCCTGA
GGAGATGAGCCAGGACAAAGGTCTCACTTGCCTGTCAAGCAG
CTGTGGTGTGTCTCTTGAGGCCTCAGTTTCCCCATTTATAAAA
CAATCAAAATTGATCTCAGATCCATCCCTCCTAGTGCTGATGT
TCCAAGACCAAAAGGCCCAGAAGAGTGGGCCTAGCTAATGTT
TCTGGGCCTCATCGTGGTAACATGCACAGCCCTTGCCACTCCC
CCATGGCCCATGTAGAAACAACCATGTGGCCTTCACTCTGCC
CACAGAAGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTT
CTGCTGAGTCAAAGCCACATGGAGGGAGGGAGCAAGGGAGA
GATGCAGAGTCATGTTTCCAGGAGGAGGTTATCTGAGCATAA
CAGGGACAGGGTGGGCCACAGGATACCTCTGAGGCTCAGGTT
CCCACCTCCACTCCACCCAAGCCTTCAATGATGCTCATGAATC
CCCAATAAGAGGAGGTGGGAGAGAACTGAGCGCAAGCAATG
GATATTGCTTAACTCAGGGGAACCAATGGCAGGCAGGCAGCT
ACCTGGGGAGTAGGCAATTCTCAGAAGTCTCTAAAGCAAATG
GTAGGACCCTATAGGAAGATAATCCAGGGGAACAGAAGGCA
GAAAGCCACCTGCCTCAAGACTCCCAACAACAGAGGGCCAC
GTGCTCCTACTTGGAGGAGGTAAGGTCCAGAGAAGTCAAGGA
ACACAGCCAAGGTCATCCAGCCAGTTGACAACCAAGCTAAGA
AGCTAAGGATCCTAGGATCATAAAAAGAATGTGTCCTGGCCG
GATGCGGTGGCTCATGCCTATAATCCAATCTTTTCACCTGTCT
CATGGGATAATGATCCAAGTTTTGCAGTGAGCCTGAAGCACC
CAGCTGAATGCCTAGCACACAGCAGGGGTCAAGGAAACAGT
GCCCTCTATGTTTGCAGCCCCCGCCCAACTCCAACTCTCTGCT
CCACGTTCCTTCCACTATTCAACACATGGCAGGAAGTCAGCC
TGTAGGGCTTCACACTACAGTCTAAGCTGACTGCCATCTGTCC
CCATCCAGGCGAGGCTGGAAACGGGGGCAGCAAAAAGCCCA
GGGACAAAGTCCCCTTCCCAGGCACATGTATTATGCACTCTG
CACCAAGGAAACCTCCAGATAAGGTCTAGAGACCAAGGGCC
ATGTACTTCGGGGGAGAGGCCAGAGGACTTCTGAGGTTTTAC
AGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGTGTAG
CCATAGTGCCGATAAGGAAAGGCCTTCAACCTGCCCTGGTCA
GCTCTTCCTGTAAGTAGAGGCCCCTTACCGAAGGCCCCAGTG
GGAGGAAGGGTCGGGAGTATTTGGGGAGAACAGCTAGATAC

ATATAGACACACACACACACACAAATACTGCTCTTGATACGA
GTTACTACAACAAGCCAGGTCC
GACCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGA
AGGGGGGTAGCCCCTTCACCAATGTAAACAAGGATGTGGGTT
CTGCGGCCACACTCTCCCCCGCCCTCCCCAGCGGCATTTCCAG
CAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCTGC
TGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAG
CTACTTGGGGAGCTATTTCTGTAGACTGAGCTTGGGGTACTG
GGAGGCAGTGCTGTTGGCGATCTGGGGGAAAACCCCCTGAGG
GTCCCATTTCTCTGCCACTGACCCCTTTGCCTGGCAGAAAGGC
TCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAAC
CTCTCTAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAGT
AACTCCTCTTCCATCTGCTGGGTGTTGTCAAAACTACACAGGA
GGTTTGCCAAGCACCTAAGGCACAGTAGGTGGTCAAGAAACA
GGAACACAATTTGTCATGACGACAATGATTCCTTCATATGCT
AGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTAT
GAAGCCCTTCCTCATTTCTGTTGCAATTCCTCAGCCAGAGGAA
AGCCCCACCACCTCTGTCCTCCAGAAAGCCCTCCTCTTCCCAG
AAGGCTGGCACACAGGGCTGGGGGCCAGGCTTGACTTCCCTG
TGGAGGGGTCTGAGGTTGGGGGGATGACCTGCAGAGGTAGG
Enh ancer GGAATTCAGAGAACTTGTTTTGATAGAAACTGAAACTCCTCC
C. T TTGCTGACGCTGCACTTGTGGGCAATCTGCACCTGTTCTCC
element 2 (Slim) CCTCCCAGAGGCCAGCTGGAAGGGGGTCCCCCACCATTCTAC
(SEQ ID NO:2) CTCTGTTCCCATCTCAAACAGGGCTCAGAAATCCTCAGAGAG
CCTGGATTATCTTCCTATAGGGTCCTACCATTTGCTTTAGAGA
CATCTGAGAACTGCCTACTCCCCAGGTAGCTGCCTGCCTCCCA
GAAGAGCCACAGCAAGTTCCGCTAAGGGCAAAAAAGGAAGC
TAGGTGTCTACAGGGAACCTAAAAACAAACCACACTAACGTG
TGTACACACAGCTGCAGAGGGAGACAGGGACCTGGTCTGCTC
ACAGCCCTTGCCACTCCCCCATGGCCCATGTAGAAACAACCA
TGTGGCCTTCACTCTGCCCACAGAAGCTAGCACCAGGACCCT
GGTCAGGGTTAGAGGTTTCTGCTGAGTCAAAGCCACATGGAG
GGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTTCCAGGAG
GAGGTTATCTGAGCATAACAGGGACAGGGTGGGCCACAGGA
TACCTCTGAGGCTCAGGTTCCCACCTCCACTCCACCCTCCAAC
TCTCTGCTCCACGTTCCTTCCACTATTCAACACATGGCAGGAA
GTCAGCCTGTAGGGCTTCACACTACAGTCTAAGCTGACTGCC
ATCTGTCCCCATCCAGGCGAGGCTGGAAACGGGGGCAGCAA
AAAGCCCAGGGACAAAGTCCCCTTCCCAGGCACATGTATTAT
GCACTCTGCACCAAGGAAACCTCCAGATAAGGTCTAGAGACC
AAGGGCCATGTACTTCGGGGGAGAGGCCAGAGGACTTCTGA
GGTTTTACAGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAA
ATGTGTAGCCATAGTGCCGATAAGGAAAGGCCTTCAACCTGC
CCTGGTCAGCTCTTCCTGTAAGTAGAGGCCCCTTACCGAAGG
CCCCAGTGGGAGGAAGGGTCGGGAGTA
1St half of Core Sub-Element 1 GACCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGA
of Enhancer AGGGGGGTAGCCCCTTCACCAATGTAAACAAGGATGTGGGTT
Element 2 CTGCGGCCACACTCTCCCCCGCCCTCCCCAGCGGCATTTCCAG
SEQ ID NO:3) CAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCTGC
( TGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAG

CTACTTGGGGAGCTATTTCTGTAGACTGAGCTTGGGGTACTG
GGAGGCAGTGCTGTTGGC
GATCTGGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCAC
TGACCCCTTTGCCTGGCAGAAAGGCTCTGCTGCCTCCTAGCTG
2nd half of Core TGTAGCAATGGATAAGGCCTTTAACCTCTCTAAGCTGAAATG
Sub-Element 1 TCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCCATCTGC
of Enhancer TGGGTGTTGTCAAAACTACACAGGAGGTTTGCCAAGCACCTA
Element 2 AGGCACAGTAGGTGGTCAAGAAACAGGAACACAATTTGTCAT
(SEQ ID NO :4) GACGACAATGATTCCTTCATATGCTAGTTACCCACGAGTCAG
CCAGAATGTTGCCATAACCACTTATGAAGCCCTTCCTCATTTC
TGTTGCAATTCCTCAGCCAGAGGAAAGCCCCA
CCACCTCTGTCCTCCAGAAAGCCCTCCTCTTCCCAGAAGGCTG
Core Sub- GCACACAGGGCTGGGGGCCAGGCTTGACTTCCCTGTGGAGGG
Element 2 of GTCTGAGGTTGGGGGGATGACCTGCAGAGGTAGGGGAATTCA
Enhancer GAGAACTTGTTTTGATAGAAACTGAAACTCCTCCTCTTGCTGA
Element 2 CGCTGCACTTGTGGGCAATCTGCACCTGTTCTCCCCTCCCAGA
(SEQ ID NO:5) GGCCAGCTGGAAGGGGGTCCCCCACCATTCTACCTCTGTTCC
CATCTCAAACAGGGCTCAGAAATCCTCA
C S b-GAGAGCCTGGATTATCTTCCTATAGGGTCCTACCATTTGCTTT
El a ore u AGAGACATCTGAGAACTGCCTACTCCCCAGGTAGCTGCCTGC
ement 3 CTCCCAGAAGAGCCACAGCAAGTTCCGCTAAGGGCAAAAAA
Enhancer GGAAGCTAGGTGTCTACAGGGAACCTAAAAACAAACCACAC
Element 2 TAACGTGTGTACACACAGCTGCAGAGGGAGACAGGGACCTG
(SEQ ID NO:6) GTCTGCT
Core -S b CACAGCCCTTGCCACTCCCCCATGGCCCATGTAGAAACAACC
u Element 4 of ATGTGGCCTTCACTCTGCCCACAGAAGCTAGCACCAGGACCC
TGGTCAGGGTTAGAGGTTTCTGCTGAGTCAAAGCCACATGGA
Enhancer GGGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTTCCAGGA
Element 2 GGAGGTTATCTGAGCATAACAGGGACAGGGTGGGCCACAGG
(SEQ ID NO:7) ATACCTCTGAGGCTCAGGTTCCCACCTCCACTCCACC
CTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCAACACATG
GCAGGAAGTCAGCCTGTAGGGCTTCACACTACAGTCTAAGCT
Core -S b GACTGCCATCTGTCCCCATCCAGGCGAGGCTGGAAACGGGGG
u Element 5 of CAGCAAAAAGCCCAGGGACAAAGTCCCCTTCCCAGGCACATG
TATTATGCACTCTGCACCAAGGAAACCTCCAGATAAGGTCTA
Enhancer GAGACCAAGGGCCATGTACTTCGGGGGAGAGGCCAGAGGAC
Element 2 TTCTGAGGTTTTACAGAGAAAAGCCAAAGGCAGCCCAGTCAG
(SEQ ID NO: 8) GGGAAATGTGTAGCCATAGTGCCGATAAGGAAAGGCCTTCAA
CCTGCCCTGGTCAGCTCTTCCTGTAAGTAGAGGCCCCTTACCG
AAGGCCCCAGTGGGAGGAAGGGTCGGGAGTA
CTAAAAGCCACCAAAACCAAGACAGCAATGAAAGTAACCT
CTGGTCTTCCTAACTGCTCATTATACACTAATTATGATGCA
Enhancer CTAGCATGCTAAGAGACACTTCCACCAGCGCCATGACAGT
element HS3 GCCATGGCAACATCAAGAAGTCACCCCACATGGTCTAAAA
(full) AGGGGAAGAACCTTCAGTTCCCGGAATTGCCCACCCCTTT
CCTGGAAAACTCATGAATAATCCACCCCTTGTTTAGCATA
(SEQ ID NO:9) TAATTAAGAAATAACTGTAAGTATCCTTAGTCCAGCAGCC
CAAGCTGCTGCTCTGCCTATGGAGTAGCCATTCTTTATTC
CTTTACTTTCTTAATAAACTTGCTTTCACATTAAAAACAAC
_____________ AACAACAACACCAACAACCAGCAAATATATCAGAGGCTTA

GCATCCCCTATTCAAGGGGACAGTTCTGGAGGCCGAGAA
AGAAGTAGAAAGGTGGAGAATAAAAGACACAAAGGAGCA
GAAGAATTTTATCTTGGCCTCCATGTGGCACCTCACAGCT
TGGGAACAACA
CTAAAAGCCACCAAAACCAAGACAGCAATGAAAGTAACCTC
Enhancer TGGTCTTCCTAACTGCTCATTATACACTAATTATGATGCACTA
element HS3 GCATGCTAAGAGACACTTCCACCAGCGCCATGACAGTGCCAT
core GGCAACATCAAGAAGTCACCCCACATGGTCTAAAAAGGGGA
(SEQ ID NO:10) AGAACCTTCAGTTCCCGGAATTGCCCACCCCTTTCCTGGAAA
ACTCATGAATAATCCACCCCTTGTTTAGCATATAAT
GGGATCTGTGTGCAGATTTACCTCTACCTACACCTGGCTGGG
GATCATAAAGAAAATCAAGGGATGCGCTACCTCCATGAATCC
AGGTTTCAGCAGAGCTAAGGGAGTGAAATTTGGGGCCATTTT
ACTTGTCTCAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCC
TTCTTTCAGCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGG
Enhancer CTGGGAAGATGAGCAATGCTTGTGAGTCACCATGATAGGAAG
element E9 (full) CAGAAGGGTCAGGAAGTCCCTGGGAGCAAGGCTTAGGGTTA
(SEQ ID NO:11) GGGTTAGGGAGAAAACACTTGGGCCTGGAGGCTCGGGGCAG
GCTTCCTAGAGGGGAGGGGTAGGAAGAGGCAGTGACAGGGC
CCACAGCAATGGAGAGGAGTTGGACTGCAGGGATGGGGTAG
GGGGACAGACGACAAGGGACACTCAGAAGACTAATGTCTGG
GAGTGGGAAAACAGTGTTTGCTCAGCCAGGAAGCTGCATCCA
GCTCTTTTATCATTTGTAGAAGACCAAGTACCCAGGCCTGGG
AGGAGCCCAGAGA
CAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCCTTCTTTCA
Enhancer GCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGGCTGGGAA
element E9 core GATGAGCAATGCTTGTGAGTCACCATGATAGGAAGCAGAAG
(SEQ ID NO:12) GGTCAGGAAGTCCCTGGGAGCAAGGCTTAGGGTTAGGGTTAG
GGAGAAAACACTTGGGCCTGGAGGCTCGGGGCAGGCTTCCTA
GAGGGGAGGGGTA
atgagtgggggcccaatgggaggaaggcccgggggccgaggagcaccagcggttcagcagaac ataccctccaccctcctccaggaccacgagaaccagcgactctttgagatgcttggacgaaaatgcttg acgctggccactgcagttgttcagctgtacctggcgctgccccctggagctgagcactggaccaagg agcattgtggggctgtgtgcttcgtgaaggataacccccagaagtcctacttcatccgcctttacggcct tcaggctggtcggctgctctgggaacaggagctgtactcacagcttgtctactccacccccaccccctt cttccacaccttcgctggagatgactgccaagcggggctgaactttgcagacgaggacgaggcccag gccttccgggccctcgtgc agg agaagatac aaaaaaggaatcag aggc aaagtggag ac ag acg ccagctacccccaccaccaacaccagccaatgaagagagaagaggagggctcccacccctgcccc tgcatccaggtggagaccaaggaggccctccagtgggtccgctctccctggggctggcgacagtgg WAS cDNA
acatccagaaccctgacatcacgagttcacgataccgtgggctcccagcacctggacctagcccagc (SEQ ID NO:13) tgataagaaacgctcagggaagaagaagatcagcaaagctgatattggtgcacccagtggattcaag catgtcagccacgtggggtgggacccccagaatggatttgacgtgaacaacctcgacccagatctgc gg agtctgttctccagggcaggaatcagcg aggcccagctcaccgacgccg agacctctaaacttat ctacgacttcattgaggaccagggtgggctggaggctgtgcggcaggagatgaggcgccaggagc c acttccgccgccc cc accgcc atctcg agg aggg aacc agctcccccggcccc ctattgtggggg gtaacaagggtcgttctggtccactgccccctgtacctttggggattgccccacccccaccaacacccc ggggacccccacccccaggccgagggggccctccaccaccaccccctccagctactggacgttctg gaccactgccccctccaccccctggagctggtgggccacccatgccaccaccaccgccaccaccgc caccgccgcccagctccgggaatggaccagcccctcccccactccctcctgctctggtgcctgccgg gggcctggcccctggtgggggtcggggagcgcttttggatcaaatccggcagggaattcagctgaac aagacccctggggccccagagagctcagcgctgcagccaccacctcagagctcagagggactggt gggggccctgatgcacgtgatgcagaagagaagcagagccatccactcctccgacgaaggggagg accaggctggcgatgaagatgaagatgatgaatgggatgactga ATGAGCGGCGGCCCCATGGGCGGCCGCCCCGGCGGCCGCGG
CGCCCCCGCCGTGCAGCAGAACATCCCCAGCACCCTGCTGCA
GGACCACGAGAACCAGCGCCTGTTCGAGATGCTGGGCCGCAA
GTGCCTGACCCTGGCCACCGCCGTGGTGCAGCTGTACCTGGC
CCTGCCCCCCGGCGCCGAGCACTGGACCAAGGAGCACTGCGG
CGCCGTGTGCTTCGTGAAGGACAACCCCCAGAAGAGCTACTT
CATCCGCCTGTACGGCCTGCAGGCCGGCCGCCTGCTGTGGGA
GCAGGAGCTGTACAGCCAGCTGGTGTACAGCACCCCCACCCC
CTTCTTCCACACCTTCGCCGGCGACGACTGCCAGGCCGGCCT
GAACTTCGCCGACGAGGACGAGGCCCAGGCCTTCCGCGCCCT
GGTGCAGGAGAAGATCCAGAAGCGCAACCAGCGCCAGAGCG
GCGACCGCCGCCAGCTGCCCCCCCCCCCCACCCCCGCCAACG
AGGAGCGCCGCGGCGGCCTGCCCCCCCTGCCCCTGCACCCCG
GCGGCGACCAGGGCGGCCCCCCCGTGGGCCCCCTGAGCCTGG
GCCTGGCCACCGTGGACATCCAGAACCCCGACATCACCAGCA
GCCGCTACCGCGGCCTGCCCGCCCCCGGCCCCAGCCCCGCCG
ACAAGAAGCGCAGCGGCAAGAAGAAGATCAGCAAGGCCGAC
Codon optimized ATCGGCGCCCCCAGCGGCTTCAAGCACGTGAGCCACGTGGGC
WAS TGGGACCCCCAGAACGGCTTCGACGTGAACAACCTGGACCCC
(SEQ ID NO:14) GACCTGCGCAGCCTGTTCAGCCGCGCCGGCATCAGCGAGGCC
CAGCTGACCGACGCCGAGACCAGCAAGCTGATCTACGACTTC
ATCGAGGACCAGGGCGGCCTGGAGGCCGTGCGCCAGGAGAT
GCGCCGCCAGGAGCCCCTGCCCCCCCCCCCCCCCCCCAGCCG
CGGCGGCAACCAGCTGCCCCGCCCCCCCATCGTGGGCGGCAA
CAAGGGCCGCAGCGGCCCCCTGCCCCCCGTGCCCCTGGGCAT
CGCCCCCCCCCCCCCCACCCCCCGCGGCCCCCCCCCCCCCGGC
CGCGGCGGCCCCCCCCCCCCCCCCCCCCCCGCCACCGGCCGC
AGCGGCCCCCTGCCCCCCCCCCCCCCCGGCGCCGGCGGCCCC
CCCATGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAGCA
GCGGCAACGGCCCCGCCCCCCCCCCCCTGCCCCCCGCCCTGG
TGCCCGCCGGCGGCCTGGCCCCCGGCGGCGGCCGCGGCGCCC
TGCTGGACCAGATCCGCCAGGGCATCCAGCTGAACAAGACCC
CCGGCGCCCCCGAGAGCAGCGCCCTGCAGCCCCCCCCCCAGA
GCAGCGAGGGCCTGGTGGGCGCCCTGATGCACGTGATGCAGA
AGCGCAGCCGCGCCATCCACAGCAGCGACGAGGGCGAGGAC
CAGGCCGGCGACGAGGACGAGGACGACGAGTGGGACGACTA
A
Full sequence of WAS Vec lentiviral vector transfer plasmid with codon optimized WASp open reading frame: (sequence is beginning from the CMV) (SEQ ID NO:29)

[0159] In various embodiments, the lentiviral vectors (LVs) described herein can have various "safety" features that can include, for example, the presence of an insulator (e.g., an FB insulator in the 3'LTR). Additionally, or alternatively, in certain embodiments, the HIV LTR has been substituted with an alternative promoter (e.g., a CMV) to yield a higher .. titer vector without the inclusion of the HIV TAT protein during packaging.
Other strong promoters (e.g., RSV, and the like can also be used).

[0160] As noted above, in various embodiments the lentiviral vectors described herein contain any one or more of the elements typically found in lentiviral vectors. Such elements include, but need not be limited to a iv region vector genome packaging signal, a Rev Responsive Element (RRE), a polypurine tract (e.g., a central polypurine tract, a 3' polypurine tract, etc.), a post-translational regulatory element (e.g., a modified Woodchuck Post-transcriptional Regulatory Element (WPRE)), an insulator, and the like, e.g., as described below.

[0161] In various embodiments the vector is a SIN vector substantially incapable of reconstituting a wild-type lentivirus through recombination.

[0162] In various embodiments the vectors described herein shows high expression in MEG-01 cells (megakaryocyte cell line), and/or in Jurkat cells (T-cell line), and/or in RAMOs cells (B-cell line). In certain embodiments the vectors described herein show high expression in CB CD34+ differentiated megakaryocytes including "pro-megakaryocytes", megakaryocytes, and platelets.

[0163] As shown above, in Example 1, the vectors described herein are believed to be effective to transduce cells at high titer and to also provide high levels of expression of a nucleic acid encoding WASp protein.

[0164] In view of these results, it is believed that LVs described herein, e.g., recombinant TAT-independent, SIN LVs that express a nucleic acid encoding a WASP can be used to effectively treat Wiskott-Aldrich Syndrome (WAS) in subjects (e.g., human and non-human mammals). It is believed these vectors can be used for the modification of stem cells (e.g., hematopoietic stem and progenitor cells) that can be introduced into a subject in need thereof for the treatment of, e.g., subjects identified as having WAS.
Moreover, it is believed that the resulting cells will produce enough of the transgenic WASp protein to demonstrate significant improvement in subject health. It is also believed the vectors can be directly administered to a subject to achieve in vivo transduction of the target (e.g., hematopoietic stem or progenitor cells) and thereby also effect a treatment of subjects in need thereof.

[0165] As noted above, in various embodiments the LVs described herein can comprise various safety features. For example, the HIV LTR has been substituted with a CMV promoter to yield higher titer vector without the inclusion of the HIV TAT
protein during packaging. In certain embodiments an insulator (e.g., the FB insulator) can be introduced into the 3'LTR for safety. The LVs are also constructed to provide efficient transduction and high titer.

[0166] It will be appreciated that the foregoing elements are illustrative and need not be limiting. In view of the teachings provided herein, suitable substitutions for these elements will be recognized by one of skill in the art and are contemplated within the scope of the teachings provided herein.
WAS codon optimization.

[0167] As noted above, in various embodiments the lentiviral vector can comprise a WAS gene or cDNA. However, in certain embodiments the nucleic acid encoding WASp is codon optimized. Numerous methods of codon optimization are known to those of skill in the art. One illustrative method is JCat (Java Codon Adaptation Tool). The jCAT tool adapts gene codon usage to most sequenced prokaryotes and various eukaryotic gene expression hosts. In contrast to many tools, JCat does not require the manual definition of highly expressed genes and is, therefore, a very rapid and easy method. Further options of JCat for codon adaptation include the avoidance of unwanted cleavage sites for restriction enzymes and Rho-independent transcription terminators. The output of JCat is both graphically and as Codon Adaptation Index (CAI) values given for the input sequence and the newly adapted sequence. JCat optimization is described by Grote et al. (2005) Nucleic Acids Res. 33(suppl 2): W526¨W531) and a JCat tool is available online at wwwjcat.de.

[0168] Another codon optimization tool is provided by GeneArt (from ThermoFisher Scientific .

[0169] Still another codon optimization tool is IDT. The IDT codon optimization tool was developed to optimize a DNA or protein sequence from one organism for expression in another by reassigning codon usage based on the frequencies of each codon's usage in the new organism. For example, valine is encoded by 4 different codons (GUG, GUU, GUC, and GUA). In human cell lines, however, the GUG codon is preferentially used (46%
use vs. 18, 24, and 12%, respectively). The codon optimization tool takes this information into account and assigns valine codons with those same frequencies. In addition, the tool algorithm eliminates codons with less than 10% frequency and re-normalizes the remaining frequencies to 100%. Moreover, the optimization tool reduces complexities that can interfere with manufacturing and downstream expression, such as repeats, hairpins, and extreme GC
content. The IDT optimization tool is available from IDT (Integrated DNA
Technologies, Coralville, Iowa) and can be found at ww.idtdna.com/CodonOpt.

[0170] Other codon optimization tools include, but are not limited to CodonW an open source software program that can be found at codonw.sourceforge.net, and the OptimumGeneTM algorithm from GenScript.

[0171] These codon optimizations are illustrative and non-limiting. Using the teaching provided here and in Example 1, the WAS codon usage can readily be optimized for particular applications.
TAT-Independent and Self inactivating lentiviral vectors.

[0172] To further improve safety, in various embodiments, the lentiviral vectors described herein comprise a TAT-independent, self-inactivating (SIN) configuration. Thus, in various embodiments it is desirable to employ in the LVs described herein an LTR region that has reduced promoter activity relative to wild-type LTR. Such constructs can be provided that are effectively "self-inactivating" (SIN) which provides a biosafety feature.
SIN vectors are ones in which the production of full-length vector RNA in transduced cells is greatly reduced or abolished altogether. This feature minimizes the risk that replication-competent recombinants (RCRs) will emerge. Furthermore, it reduces the risk that that cellular coding sequences located adjacent to the vector integration site will be aberrantly expressed.

[0173] Furthermore, a SIN design reduces the possibility of interference between the LTR and the promoter that is driving the expression of the transgene. SIN LVs can often permit full activity of the internal promoter.

[0174] The SIN design increases the biosafety of the LVs. The majority of the HIV
LTR is comprised of the U3 sequences. The U3 region contains the enhancer and promoter elements that modulate basal and induced expression of the HIV genome in infected cells and in response to cell activation. Several of these promoter elements are essential for viral replication. Some of the enhancer elements are highly conserved among viral isolates and have been implicated as critical virulence factors in viral pathogenesis. The enhancer elements may act to influence replication rates in the different cellular target of the virus

[0175] As viral transcription starts at the 3 end of the U3 region of the 5' LTR, those sequences are not part of the viral mRNA and a copy thereof from the 3' LTR
acts as template for the generation of both LTRs in the integrated provirus. If the 3' copy of the U3 region is altered in a retroviral vector construct, the vector RNA is still produced from the intact 5' LTR in producer cells, but cannot be regenerated in target cells. Transduction of such a vector results in the inactivation of both LTRs in the progeny virus. Thus, the retrovirus is self-inactivating (SIN) and those vectors are known as SIN transfer vectors.

[0176] In certain embodiments self-inactivation is achieved through the introduction of a deletion in the U3 region of the 3' LTR of the vector DNA, i.e., the DNA
used to produce the vector RNA. During RT, this deletion is transferred to the 5' LTR of the proviral DNA.
Typically, it is desirable to eliminate enough of the U3 sequence to greatly diminish or abolish altogether the transcriptional activity of the LTR, thereby greatly diminishing or abolishing the production of full-length vector RNA in transduced cells.
However, it is generally desirable to retain those elements of the LTR that are involved in polyadenylation of the viral RNA, a function typically spread out over U3, R and U5.
Accordingly, in certain embodiments, it is desirable to eliminate as many of the transcriptionally important motifs from the LTR as possible while sparing the polyadenylation determinants.

[0177] The SIN design is described in detail in Zufferey et al. (1998) J Virol. 72(12):
9873-9880, and in U.S. Patent No: 5,994,136. As described therein, there are, however, limits to the extent of the deletion at the 3' LTR. First, the 5' end of the U3 region serves another essential function in vector transfer, being required for integration (terminal dinucleotide+att sequence). Thus, the terminal dinucleotide and the att sequence may represent the 5' boundary of the U3 sequences which can be deleted. In addition, some loosely defined regions may influence the activity of the downstream polyadenylation site in the R region. Excessive deletion of U3 sequence from the 3'LTR may decrease polyadenylation of vector transcripts with adverse consequences both on the titer of the vector in producer cells and the transgene expression in target cells.

[0178] Additional SIN designs are described in U.S. Patent Publication No:
2003/0039636. As described therein, in certain embodiments, the lentiviral sequences removed from the LTRs are replaced with comparable sequences from a non-lentiviral retrovirus, thereby forming hybrid LTRs. In particular, the lentiviral R
region within the LTR can be replaced in whole or in part by the R region from a non-lentiviral retrovirus. In certain embodiments, the lentiviral TAR sequence, a sequence which interacts with TAT
protein to enhance viral replication, is removed, preferably in whole, from the R region. The TAR sequence is then replaced with a comparable portion of the R region from a non-lentiviral retrovirus, thereby forming a hybrid R region. The LTRs can be further modified to remove and/or replace with non-lentiviral sequences all or a portion of the lentiviral U3 and U5 regions.

[0179] Accordingly, in certain embodiments, the SIN configuration provides a retroviral LTR comprising a hybrid lentiviral R region that lacks all or a portion of its TAR
sequence, thereby eliminating any possible activation by TAT, wherein the TAR
sequence or portion thereof is replaced by a comparable portion of the R region from a non-lentiviral retrovirus, thereby forming a hybrid R region. In a particular embodiment, the retroviral LTR
comprises a hybrid R region, wherein the hybrid R region comprises a portion of the HIV R
region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID
NO: 10 in US 2003/0039636) lacking the TAR sequence, and a portion of the MoMSV R
region (e.g., a portion comprising or consisting of the nucleotide sequence shown in SEQ ID
NO: 9 in 2003/0039636) comparable to the TAR sequence lacking from the HIV R
region.
In another particular embodiment, the entire hybrid R region comprises or consists of the nucleotide sequence shown in SEQ ID NO: 11 in 2003/0039636.

[0180] Suitable lentiviruses from which the R region can be derived include, for example, HIV (HIV-1 and HIV-2), EIV, SIV and FIV. Suitable retroviruses from which non-lentiviral sequences can be derived include, for example, MoMSV, MoMLV, Friend, MSCV, RSV and Spumaviruses. In one illustrative embodiment, the lentivirus is HIV
and the non-lentiviral retrovirus is MoMSV.

[0181] In another embodiment described in US 2003/0039636, the LTR
comprising a hybrid R region is a left (5') LTR and further comprises a promoter sequence upstream from the hybrid R region. Preferred promoters are non-lentiviral in origin and include, for example, the U3 region from a non-lentiviral retrovirus (e.g., the MoMSV U3 region). In one particular embodiment, the U3 region comprises the nucleotide sequence shown in SEQ ID
NO: 12 in US 2003/0039636. In another embodiment, the left (5') LTR further comprises a lentiviral U5 region downstream from the hybrid R region. In one embodiment, the U5 region is the HIV U5 region including the HIV att site necessary for genomic integration. In another embodiment, the U5 region comprises the nucleotide sequence shown in SEQ ID
NO: 13 in US 2003/0039636. In yet another embodiment, the entire left (5') hybrid LTR
comprises the nucleotide sequence shown in SEQ ID NO: 1 in US 2003/0039636.

[0182] In another illustrative embodiment, the LTR comprising a hybrid R region is a right (3') LTR and further comprises a modified (e.g., truncated) lentiviral U3 region upstream from the hybrid R region. The modified lentiviral U3 region can include the att sequence, but lack any sequences having promoter activity, thereby causing the vector to be .. SIN in that viral transcription cannot go beyond the first round of replication following chromosomal integration. In a particular embodiment, the modified lentiviral U3 region upstream from the hybrid R region consists of the 3 end of a lentiviral (e.g., HIV) U3 region up to and including the lentiviral U3 att site. In one embodiment, the U3 region comprises the nucleotide sequence shown in SEQ ID NO: 15 in US 2003/0039636. In another embodiment, the right (3') LTR further comprises a polyadenylation sequence downstream from the hybrid R region. In another embodiment, the polyadenylation sequence comprises the nucleotide sequence shown in SEQ ID NO: 16 in US 2003/0039636. In yet another embodiment, the entire right (5') LTR comprises the nucleotide sequence shown in SEQ ID
NO: 2 or 17 of US 2003/0039636.

[0183] Thus, in the case of HIV based LV, it has been discovered that such vectors tolerate significant U3 deletions, including the removal of the LTR TATA box (e.g., deletions from -418 to -18), without significant reductions in vector titers. These deletions render the LTR region substantially transcriptionally inactive in that the transcriptional ability of the LTR in reduced to about 90% or lower.

[0184] It has also been demonstrated that the trans-acting function of Tat becomes dispensable if part of the upstream LTR in the transfer vector construct is replaced by constitutively active promoter sequences (see, e.g., Dull et al. (1998) J
Virol. 72(11): 8463-8471. Furthermore, we show that the expression of rev in trans allows the production of high-titer HIV-derived vector stocks from a packaging construct which contains only gag and pol. This design makes the expression of the packaging functions conditional on complementation available only in producer cells. The resulting gene delivery system, conserves only three of the nine genes of HIV-1 and relies on four separate transcriptional units for the production of transducing particles.

[0185] In one embodiments illustrated in Example 1, the cassette expressing a nucleic acid encoding WASp is a SIN vector with the CMV enhancer/promoter substituted in the 5' LTR.

[0186] It will be recognized that the CMV promoter typically provides a high level of non-tissue specific expression. Other promoters with similar constitutive activity include, but are not limited to the RSV promoter, and the SV40 promoter. Mammalian promoters such as the beta-actin promoter, ubiquitin C promoter, elongation factor lapromoter, tubulin promoter, etc., may also be used.

[0187] The foregoing SIN configurations are illustrative and non-limiting. Numerous SIN configurations are known to those of skill in the art. As indicated above, in certain embodiments, the LTR transcription is reduced by about 95% to about 99%. In certain embodiments LTR may be rendered at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95% at least about 96%, at least about 97%, at least about 98%, or at least about 99% transcriptionally inactive.
Insulator element

[0188] In certain embodiments, to further enhance biosafety, insulators are inserted into the lentiviral vectors described herein. Insulators are DNA sequence elements present throughout the genome. They bind proteins that modify chromatin and alter regional gene expression. The placement of insulators in the vectors described herein offer various potential benefits including, inter alia: 1) Shielding of the vector from positional effect variegation of expression by flanking chromosomes (i.e., barrier activity);
and 2) Shielding flanking chromosomes from insertional trans-activation of gene expression by the vector (enhancer blocking). Thus, insulators can help to preserve the independent function of genes or transcription units embedded in a genome or genetic context in which their expression may otherwise be influenced by regulatory signals within the genome or genetic context (see, e.g., Burgess-Beusse et al. (2002) Proc. Natl. Acad. Sci. USA, 99: 16433; and Zhan et al. (2001) Hum. Genet., 109: 471). In the present context insulators may contribute to protecting lentivirus-expressed sequences from integration site effects, which may be mediated by cis-acting elements present in genomic DNA and lead to deregulated expression of transferred sequences. In various embodiments LVs are provided in which an insulator sequence is inserted into one or both LTRs or elsewhere in the region of the vector that integrates into the cellular genome.

[0189] The first and best characterized vertebrate chromatin insulator is located within the chicken 0-globin locus control region. This element, which contains a DNase-I
hypersensitive site-4 (cHS4), appears to constitute the 5' boundary of the chicken 0-globin locus (Prioleau et al. (1999) EMBO J. 18: 4035-4048). A 1.2-kb fragment containing the cHS4 element displays classic insulator activities, including the ability to block the interaction of globin gene promoters and enhancers in cell lines (Chung et al.
(1993) Cell, 74:

505-514), and the ability to protect expression cassettes in Drosophila (Id.), transformed cell lines (Pikaart et al. (1998) Genes Dev. 12: 2852-2862), and transgenic mammals (Wang et al.
(1997) Nat. Biotechnol., 15: 239-243; Taboit-Dameron et al. (1999) Transgenic Res., 8: 223-235) from position effects. Much of this activity is contained in a 250-bp fragment. Within this stretch is a 49-bp cHS4 core (Chung et al. (1997) Proc. Natl. Acad. Sci., USA, 94: 575-580) that interacts with the zinc finger DNA binding protein CTCF implicated in enhancer-blocking assays (Bell et al. (1999) Cell, 98: 387-396).

[0190] One illustrative and suitable insulator is Hi (FII/BEAD-A), a 77 bp insulator element, that contains the minimal CTCF binding site enhancer-blocking components of the .. chicken 0-globin 5 HS4 insulators and a homologous region from the human T-cell receptor alpha/delta blocking element alpha/delta I (BEAD-I) insulator described by Ramezani et al.
(2008) Stem Cell 26: 3257-3266. The FB "synthetic" insulator has full enhancer blocking activity. This insulator is illustrative and non-limiting. Other suitable insulators may be used including, for example, the full-length chicken beta-globin HS4 or insulator sub-fragments thereof, the ankyrin gene insulator, and other synthetic insulator elements.
Packaging signal.

[0191] In various embodiments the vectors described herein further comprise a packaging signal. A "packaging signal," "packaging sequence," or "PSI
sequence" is any nucleic acid sequence sufficient to direct packaging of a nucleic acid whose sequence comprises the packaging signal into a retroviral particle. The term includes naturally occurring packaging sequences and also engineered variants thereof. Packaging signals of a number of different retroviruses, including lentiviruses, are known in the art. One illustrative, but non-limiting PSI is provided by SEQ ID NO:21.
Rev Responsive Element (RRE).

[0192] In certain embodiments the lentiviral vectors described herein comprise a Rev response element (RRE) to enhance nuclear export of unspliced RNA. RREs are well known to those of skill in the art. Illustrative RREs include, but are not limited to RREs such as that located at positions 7622-8459 in the HIV NL4-3 genome (Genbank accession number AF003887) as well as RREs from other strains of HIV or other retroviruses.
Such sequences are readily available from Genbank or from the database with URL hiv-web.lanl.gov/content/index. One illustrative, but non-limiting RRE is shown in SEQ ID
NO:22).

PolyPurine Tract (cPPT, 3'PPT).

[0193] In various embodiments the lentiviral vectors described herein further include a polypurine tract (e.g., central polypurine tract (cPPT), 3 poplypurine tract (3'PPT)).
Insertion of a fragment containing the 3'PPT (see, e.g., SEQ ID NO:24) or the central polypurine tract (cPPT) in lentiviral (e.g., HIV-1) vector constructs is known to enhance transduction efficiency.
Expression-Stimulating Posttranscriptional Regulatory Element (PRE)

[0194] In certain embodiments the lentiviral vectors (LVs) described herein may comprise any of a variety of posttranscriptional regulatory elements (PREs) whose presence within a transcript increases expression of the heterologous nucleic acid (e.g., a nucleic acid that encodes WASp) at the protein level. PREs may be particularly useful in certain embodiments, especially those that involve lentiviral constructs with modest promoters.

[0195] One type of PRE is an intron positioned within the expression cassette, which can stimulate gene expression. However, introns can be spliced out during the life cycle events of a lentivirus. Hence, if introns are used as PREs they are typically placed in an opposite orientation to the vector genomic transcript.

[0196] Posttranscriptional regulatory elements that do not rely on splicing events offer the advantage of not being removed during the viral life cycle. Some examples are the posttranscriptional processing element of herpes simplex virus, the posttranscriptional regulatory element of the hepatitis B virus (HPRE) and the woodchuck hepatitis virus (WPRE). Of these the WPRE is typically preferred as it contains an additional cis-acting element not found in the HPRE. This regulatory element is typically positioned within the vector so as to be included in the RNA transcript of the transgene, but outside of stop codon of the transgene translational unit.

[0197] The WPRE is characterized and described in U.S. Pat. No: 6,136,597.
As described therein, the WPRE is an RNA export element that mediates efficient transport of RNA from the nucleus to the cytoplasm. It enhances the expression of transgenes by insertion of a cis-acting nucleic acid sequence, such that the element and the transgene are contained within a single transcript. Presence of the WPRE in the sense orientation was shown to increase transgene expression by up to 7- to 10-fold. Retroviral vectors transfer sequences in the form of cDNAs instead of complete intron-containing genes as introns are generally spliced out during the sequence of events leading to the formation of the retroviral particle. Introns mediate the interaction of primary transcripts with the splicing machinery.

Because the processing of RNAs by the splicing machinery facilitates their cytoplasmic export, due to a coupling between the splicing and transport machineries, cDNAs are often inefficiently expressed. Thus, the inclusion of the WPRE (see, e.g., SEQ ID
NO:23) in a vector results in enhanced expression of transgenes.
Transduced Host Cells and Methods of cell transduction.

[0198] The recombinant lentiviral vectors (LV) and resulting virus described herein are capable of transferring a heterologous nucleic acid sequence (e.g., a nucleic acid encoding WASp) into a mammalian cell. In various embodiments, for delivery to cells, vectors described herein are preferably used in conjunction with a suitable packaging cell line or co-transfected into cells in vitro along with other vector plasmids containing the necessary retroviral genes (e.g., gag and poll to form replication incompetent virions capable of packaging the vectors of the present invention and infecting cells.

[0199] In certain embodiments the vectors are introduced via transfection into a packaging cell line. The packaging cell line produces viral particles that contain the vector genome. Methods for transfection are well known by those of skill in the art.
After cotransfection of the packaging vectors and the transfer vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art. Thus, the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with or without a dominant selectable marker, such as neomycin, DHFR, Glutamine synthetase, followed by selection in the presence of the appropriate drug and isolation of clones. In certain embodiments the selectable marker gene can be linked physically to the packaging genes in the construct.

[0200] Stable cell lines wherein the packaging functions are configured to be expressed by a suitable packaging cell are known (see, e.g., U.S. Patent No.
5,686,279, which describes packaging cells). In general, for the production of virus particles, one may employ any cell that is compatible with the expression of lentiviral Gag and Pol genes, or any cell that can be engineered to support such expression. For example, producer cells such as 293T
cells and HT1080 cells may be used.

[0201] The packaging cells with a lentiviral vector incorporated therein form producer cells. Producer cells are thus cells or cell-lines that can produce or release packaged infectious viral particles carrying the therapeutic gene of interest (e.g., nucleic acid encoding WASp). These cells can further be anchorage dependent which means that these cells will grow, survive, or maintain function optimally when attached to a surface such as glass or plastic. Some examples of anchorage dependent cell lines used as lentiviral vector packaging cell lines when the vector is replication competent are HeLa or 293 cells and PERC.6 cells.

[0202] Accordingly, in certain embodiments, methods are provided of delivering a gene to a cell which is then integrated into the genome of the cell, comprising contacting the cell with a virion containing a lentiviral vector described herein. The cell (e.g., in the form of tissue or an organ) can be contacted (e.g., infected) with the virion ex vivo and then delivered to a subject (e.g., a mammal, animal or human) in which the gene (e.g., a nucleic acid encoding WASp) will be expressed. In various embodiments the cell can be autologous to the subject (i.e., from the subject) or it can be non-autologous (i.e., allogeneic or xenogenic) to the subject. Moreover, because the vectors described herein are capable of being delivered to both dividing and non-dividing cells, the cells can be from a wide variety including, for example, bone marrow cells, mesenchymal stem cells (e.g., obtained from adipose tissue), and other primary cells derived from human and animal sources. Alternatively, the virion can be directly administered in vivo to a subject or a localized area of a subject (e.g., bone marrow).

[0203] In certain embodiments, the lentivectors described herein will be particularly useful in the transduction of human hematopoietic progenitor cells or a hematopoietic stem cells, obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T
lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+
hematopoetic stem and progenitor cells.
Gene therapy.

[0204] In still other embodiments, methods are provided for transducing a human hematopoietic stem cell. In certain embodiments the methods involve contacting a population of human cells that include hematopoietic stem cells with one of the foregoing lentivectors under conditions to effect the transduction of a human hematopoietic progenitor cell in said population by the vector. The stem cells may be transduced in vivo or in vitro, depending on the ultimate application. Even in the context of human gene therapy, such as gene therapy of human stem cells, one may transduce the stem cell in vivo or, alternatively, transduce in vitro followed by infusion of the transduced stem cell into a human subject. In one aspect of this embodiment, the human stem cell can be removed from a human, e.g., a WAS patient, using methods well known to those of skill in the art and transduced as noted above. The transduced stem cells are then reintroduced into the same or a different human.
Stem cell/progenitor cell gene therapy.

[0205] In various embodiments the lentivectors described herein are particularly useful for the transduction of human hematopoietic progenitor cells or haematopoietic stem cells (HSCs), obtained either from the bone marrow, the peripheral blood or the umbilical cord blood, as well as in the transduction of a CD4+ T cell, a peripheral blood B or T
lymphocyte cell, and the like. In certain embodiments particularly preferred targets are CD34+ hematopoietic stem and progenitor cells.

[0206] When cells, for instance CD34+ cells, dendritic cells, peripheral blood cells or tumor cells are transduced ex vivo, the vector particles are incubated with the cells using a dose generally in the order of between 1 to 50 multiplicities of infection (MOI) which also corresponds to 1 x 105 to 50 x 105 transducing units of the viral vector per 105 cells. This can include amounts of vector corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, and 50 MOI. Typically, the amount of vector may be expressed in terms of transducing units (TU).

[0207] In certain embodiments cell-based therapies involve providing stem cells and/or hematopoietic precursors, transduce the cells with the lentivirus encoding, e.g., a nucleic acid that encodes WASp, and then introduce the transformed cells into a subject in need thereof (e.g., a subject with a mutation in the WAS gene).

[0208] In certain embodiments the methods involve isolating population of cells, e.g., stem cells from a subject, optionally expand the cells in tissue culture, and administer the lentiviral vector whose presence within a cell results in production of a normal WASp in the cells in vitro. The cells are then returned to the subject, where, for example, they may provide a population of red blood cells that produce the WASp.

[0209] In some illustrative, but non-limiting, embodiments, a population of cells, which may be cells from a cell line or from an individual other than the subject, can be used.
Methods of isolating stem cells, immune system cells, etc., from a subject and returning them to the subject are well known in the art. Such methods are used, e.g., for bone marrow transplant, peripheral blood stem cell transplant, etc., in patients undergoing chemotherapy.

[0210] Where stem cells are to be used, it will be recognized that such cells can be derived from a number of sources including bone marrow (BM), cord blood (CB), mobilized peripheral blood stem cells (mPBSC), and the like. In certain embodiments the use of induced pluripotent stem cells (IPSCs) is contemplated. Methods of isolating hematopoietic stem cells (HSCs), transducing such cells and introducing them into a mammalian subject are well known to those of skill in the art.

[0211] In certain embodiments a lentiviral vector described herein (see, e.g., Figures 18-21 with a nucleic acid encoding WASp instead of mCit is used in stem cell gene therapy for WAS by introducing a nucleic acid that encodes WASp into the into the bone marrow stem cells of patients with WAS followed by autologous transplantation.
Direct introduction of vector.

[0212] In certain embodiments direct treatment of a subject by direct introduction of the vector(s) described herein is contemplated. The lentiviral compositions may be formulated for delivery by any available route including, but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal. Commonly used routes of delivery include inhalation, parenteral, and transmucosal.

[0213] In various embodiments pharmaceutical compositions can include an LV in combination with a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0214] In some embodiments, active agents, i.e., a lentiviral described herein and/or other agents to be administered together the vector, are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such compositions will be apparent to those skilled in the art. Suitable materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
Liposomes can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S.
Pat. No.
4,522,811. In some embodiments the composition is targeted to particular cell types or to cells that are infected by a virus. For example, compositions can be targeted using monoclonal antibodies to cell surface markers, e.g., endogenous markers or viral antigens expressed on the surface of infected cells.

[0215] It is advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit comprising a predetermined quantity of a LV calculated to produce the desired therapeutic effect in association with a pharmaceutical carrier.

[0216] A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Unit dose of the LV described herein may conveniently be described in terms of transducing units (T.U.) of lentivector, as defined by titering the vector on a cell line such as HeLa or 293. In certain embodiments unit doses can range from 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013 T.U. and higher.

[0217] Pharmaceutical compositions can be administered at various intervals and over different periods of time as required, e.g., one time per week for between about 1 to about 10 weeks; between about 2 to about 8 weeks; between about 3 to about 7 weeks;
about 4 weeks;
about 5 weeks; about 6 weeks, etc. It may be necessary to administer the therapeutic composition on an indefinite basis. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Treatment of a subject with a LV can include a single treatment or, in many cases, can include a series of treatments.

[0218] Illustrative, but non-limiting, doses for administration of gene therapy vectors and methods for determining suitable doses are known in the art. It is furthermore understood that appropriate doses of a LV may depend upon the particular recipient and the mode of administration. The appropriate dose level for any particular subject may depend upon a variety of factors including the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate: of excretion, other administered therapeutic agents, and the like.

[0219] In certain embodiments lentiviral gene therapy vectors described herein can be delivered to a subject by, for example, intravenous injection, local administration, or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci.
USA, 91: 3054). In certain embodiments vectors may be delivered orally or inhalationally and may be encapsulated or otherwise manipulated to protect them from degradation, enhance uptake into tissues or cells, etc. Pharmaceutical preparations can include a LV in an acceptable diluent, or can comprise a slow release matrix in which a LV is imbedded. Alternatively or additionally, where a vector can be produced intact from recombinant cells, as is the case for retroviral or lentiviral vectors as described herein, a pharmaceutical preparation can include one or more cells which produce vectors. Pharmaceutical compositions comprising a LV
described herein can be included in a container, pack, or dispenser, optionally together with instructions for administration.

[0220] The foregoing compositions, methods and uses are intended to be illustrative and not limiting. Using the teachings provided herein other variations on the compositions, methods and uses will be readily available to one of skill in the art.
Example 1 Development of Lentiviral Vectors for Treatment of Wiskott-Aldrich Syndrome (WAS)

[0221] The goal of the experiments described below was to develop a novel lentiviral vector driven by endogenous regulatory elements of the native WAS gene for the treatment of Wiskott-Aldrich Syndrome. In particular, it was desired to develop a vector that has higher expression than the current WAS1.6 lentiviral vector in megakaryocytes and consequently able to restore platelet counts to normal levels in WAS patients.
Additionally, it was desired to maintain at least a similar level of expression in all other hematopoietic cell lineages and to restore T, B and NK cell counts and function.

[0222] A bioinformatic analysis (using publicly available databases:
Project Encode, Ensemnbl, FANTOM, VISTA Enhancer Browser, GeneHancer) was utilized to elucidate the endogenous regulatory elements of the native WAS gene.

[0223] The WAS1.6 vector is driven by a 1600bp promoter fragment immediately upstream of the transcription start site. We identified two regulatory elements "HS1" and "1-152" within this fragment which are 417bp and 190bp, respectively, in size.
Thus, we identified 1026bp of inert sequence in the 1633bp fragment which could be removed to decrease promoter size.

[0224] We note that the 1.6kb promoter is insufficient to drive wildtype levels of expression in megakaryocytes and as a result patients remain microthrombocytopenic. In view of this we hypothesized that additional endogenous enhancer elements are necessary to drive wildtype levels of expression of WAS in megakaryocytes.

[0225] A proximal analysis of the WAS gene identified new enhancer elements "HS4"
and "HS3" that contain megakaryocyte DNaseI HS sites and that may act as enhancers to boost expression in megakaryocytes. A series of lentiviral vectors (LVs) containing various combinations of these regions and a reporter gene were created to evaluate activity of these regulatory elements (see, e.g., Figure 1, panel A). Additionally the E3 element (Thurman et al. (2012) Nature, 489: 75-82), proposed to be one of the strongest enhancers in the human genome has megakaryocyte hypersensitivity. Accordingly, we also constructed a vector to evaluate E3 enhancer activity in this context (see, e.g., Figure 1, panel B).

[0226] MEG-01 cells (megakaryocyte cell lines) were transduced with the WAS
vectors in order to determine if any of the newly identified enhancer elements increases expression in megakaryocyte lineage. The cells were cultured for 14 days and flow cytometry was performed for expression (mCitrine) and VCN analysis.

[0227] As shown in Figure 2 the HS1 minimal promoter (417bp) appears to be the main driver of expression in the 1.6kb vector in megakaryocytes. Additionally, H52 seems to be inert in megakaryocyte cell lines, however this element may have enhancer activity in other lineages. H53 appears to give a major boost in expression in megakaryocyte cell lines and H54 also appears to give a boost in expression in megakaryocyte cell lines. There is an effect of combining H53 and H54 and E3 is a strong non-lineage specific enhance that is also active in megakaryocytes.

[0228] The clinical 1.6kb vector is shown to express at curative levels in all hematopoietic cell lines except for megakaryocytes (patients are no longer immune deficient but still thrombocytopenic). As we show, an illustrative slim version of WAS1.6 is H52-HS1 since H52-HS1 contains all the regulatory regions WAS1.6 has (while saving 1.0 kb of sequence) suggesting that this can be used as a vector backbone. Adding the novel H53 and H54 fragment has been shown to boost expression in megakaryocytes therefore HS2-HS1 may provide therapeutic levels of expression in all hematopoietic cell lineages.
One illustrative suitable vector (H54-H53-H52-HS1) is only 155bp larger than WAS1.6, but was shown to have ¨2-fold higher expression in megakaryocytes.

[0229] However, it remained to be determined if f152-HS 1pro performs the same as WAS1.6 in other cell lineages (T cells and B cells). Additionally, we wished to determine if H53 and H54 further boost expression in other hematopoietic cell lineages, and if the H52 enhancer element is necessary. In this regard we recognized that H52 enhancer may have enhancer functions in other cell types or may be completely inert.
Additionally, we wanted to determine if HS 1pro could perform the same as WAS1.6 and if adding H54 and H53 to H52-HS1 negatively affects expression in other cell types?

[0230] To address these questions, the WAS vectors were transduced into Jurkats (T-cell line) and RAMOs (B-cell line). The cells were cultured for 14 days and expression was analyzed by flow cytometry and VCN.

[0231] Expression levels of the WAS vectors in Jurkat cells are shown in Figure 3 and the expression levels in RAMOs cells are show in Figure 4. As illustrated by these figures, the bare minimal promoter HS 1pro (SEQ NO:1) functions the same as WAS1.6 in all cell lines test (MEG-01, CMK, Jurkats and RAMOs). The H52 enhancer, by itself, seems to be relatively inert (190bp). By eliminating the inert 1 kb sequence and the 190bp H52 sequence we can save up to about 1.2kb of sequence.

[0232] The H53 enhancer appears to increase expression in megakaryocyte cell lines, but is inert in B-cells and T-cells. The H54 enhancer appears to increase expression in megakaryocytes and B-cells but is inert in T-cells. The E3 enhancer appears to increase expression in megakaryocytes but is inert in B and T cells (non-endogenous element).

[0233] In view of these discoveries, one suitable WAS vector comprises H52-HS1pro. This vector is only 200bp larger than WAS1.6, but provides higher expression than WAS1.6 in megakaryocytes, and B-cells and a similar level of expression compared to WAS1.6 in T-cells.

[0234] We proceed to reanalyze the WAS locus in depth and identified 13 putative endogenous enhancer elements contained within a 1.1 million base pair window spanning 850kb upstream and 250kb downstream of the WAS gene. Three of the elements were previously identified in our proximal bioinformatic analysis of the WAS locus.
In order to experimentally identify the critical enhancer elements that regulate the WAS
gene, each putative enhancer element was cloned upstream of the endogenous minimal WAS
promoter (HS 1pro) to drive expression of mCitrine (see, Figure 5, panel A). The expression of these vectors was compared to the gamma-retroviral vector (CMMP-mCit, see, Figure 5, panel B) used in previous controls and which was able to restore platelet counts to normal levels.

[0235] Cells were transduced with the vectors, cultured for 14 days and expression was analyzed by flow cytometry and VCN. Figure 6 shows the data for MEG-01 (megakaryoblast cell line), Figure 7 shows the data for Jurkat cells (T-cell line), and Figure 8 shows the results or RAMOs cells (B-cell line). As shown in these Figures, all 10 newly identified enhancer elements increase expression in MEG-01 cells (Megakaryoblastic cell line) at levels higher than the WAS1.6 vector and our previous lentiviral vector (HS4,3,2,1).
Elements 1, 3, 7, 8 and 9 each independently drive expression higher than the previous clinical y-retroviral CMMP-mCit vector. All vectors express higher than the y-retroviral vector and at similar levels to WAS1.6 in Jurkats (T-cell line), and all vectors express higher than the y -retroviral vector and at similar levels to WAS1.6 in RAMOS (B-cell line).
Additionally, element 2 increases expression over 2 fold higher compared to WAS1.6 and CMMP-mCit.

[0236] The 10 elements were screened again in cord blood (CB) CD34+
HSPCs differentiated into megakaryocytes and platelets to confirm result from the cell lines. Figures 9-11 show expression levels of the WAS vectors in pro-megakaryocytes, megakaryocytes, and platelets, respectively. As shown in these figures, the data from CB CD34+
differentiated megakaryocytes differ from data from MEG-01 cells (megakaryoblast cell line). In particular, only enhancer element 2 seems to boost expression in the CB CD34+
megakaryocytes. While this construct expresses 2-fold higher than WAS 1.6, it under expresses compared to the y-retroviral construct.

[0237] One proposed lead vector was to comprise XXX-H52-HS1-WASp-WPRE
where XXX represents additional enhancer elements that can be added. The HS2-component comprise the two functional elements within the WAS1.6 promoter where HS1 is the main driver and H52 provides an extra 190bp. Various constructs comprising these elements and enhancer elements 1-10 were constructed and evaluated in pro-megakaryocytes (Figure 12), megakaryocytes (Figure 13), and platelets (Figure 14).

[0238] It was determined that optimal vectors could include various components of one or more of enhancer regions H53, E2, E9, and E10. It was noted that the H53 is 531bp, E2 is 3678bp, E9 is 555 bp, and El is 455bp. Particularly in view of the length of enhancer region E2, it was desirable to identified smaller effective fragments of these regions. An analysis of enhancer element 2 (260kb downstream of WAS) was found to contain fragments approximating about 3.7 kb. The 5 E2 fragments: E2-1 (first half of core sub-element 1 + second half of core sub-element 1, see, e.g., Table 1, SEQ ID
NOs:3 and 4), respectively), E2-2 (see, e.g., Table 1, SEQ ID NO:5) , E2-3 (see, e.g., Table 1, SEQ ID
NO:6), E2-4 (see, e.g., Table 1, SEQ ID NO:7), and E2-5 (see, e.g., Table 1, SEQ ID NO:8) were cloned into 5 different vectors so that the most active fragments of E2 could be identified for inclusion in the vector. The expression of these vectors in in pro-megakaryocytes (Figure 15), megakaryocytes (Figure 16), and platelets (Figure 17) was determined. As shown in Figures 15-17, no one element of E2 seems to be the main driver of expression. Sub-elements 2 and 3 of E2 seem not to have any effect on expression when tested alone. E2 is a driver of megakaryocyte expression but desirably can be slimmed down.
This enhancer provides 3 fold higher expression than WAS1.6 in platelets, and 1.6 fold higher expression than WAS1.6 in megakaryocytes. However, the E2 enhancer under expresses compared to the y-retroviral vector, but the increase compared to WAS1.6 may be sufficient to increase platelet counts to normal levels. There is a discrepancy of expression of enhancer elements between CB CD34+ differentiated megakaryocytes and megakaryocyte cell line (MEG-01 cells) as elements 1, 2, 3, 7, 8 and 9 each independently drive expression higher than the previous clinical y-retroviral CMMP-mCit vector in MEG-01 cells, but all under express compare to CMMP-mCit in CB CD34+ megakaryocytes.

[0239] In view of these observations certain particular suitable vectors include, but are not limited to:
1) E2(all slim)-HS 1pro-mCit-WPRE (5.6kb);
2) E9(slim)-HS3(slim)-E2(all slim)-HS 1pro-mCit-WPRE (6.1kb);
3) E9(slim)-HS3(slim)-1,4,5(slim)of E2-HS 1pro-mCit-WPRE (5.6kb); and 4) E9(slim)-H53(slim)-1st half of 1(slim) and 5(slim) of E2-HS 1pro-mCit-WPRE (5.0kb).

[0240] The sizes listed above are with the mCit reporter in the open reading frame.
Sizes will differ with a nucleic acid encoding Wasp in the open reading frame.

[0241] It is noted that vector (3) above eliminates sub-elements 2 and 3 in Enhancer element 2, while vector (4) above eliminates the second half of sub-element 1, and sub-elements 2, 3, and 4 of Enhancer element 2.

[0242] It will also be recognized that for clinical use (e.g., to treat WAS) the mCitrine open reading frame will be replaced with a nucleic acid encoding the WASp protein.
Illustrative nucleic acids include, but are not limited to a WAS cDNA, and a codon-optimized WAS nucleic acid.
Example 2 Identification of Lead Candidate Vector

[0243] Example 1, above, described the generation of four lead candidate vectors: 1) E2(all slim)-HS 1pro-mCit-WRPE; 2) E9(slim)-H53(slim)-E2(all slim)-HS 1pro-mCit-WRPE;
3) E9(slim)-H53(slim)-1,4,5(slim) of E2-HS 1pro-mCit-WRPE; and 4) E9(slim)-H53(slim)-1st half of 1 (slim) and 5 (slim) of E2-HS 1pro-mCit-WRPE.

[0244] This example describes the screening of these vectors in CB
CD34+ HSPC
differentiation megakaryocytes and platelets to determine a lead candidate vector.
Additionally, codon optimization of WASp to replace the open reading frame of mCitrine with WASp and to express the actual therapeutic protein was evaluated.
Correction of WASp expression in WAS patient T and B cell lines is also demonstrated.

[0245] In order to determine a lead candidate vector, we screened the 4 candidate vectors in megakaryocytes and platelets differentiated from healthy donor (HD) cord blood (CB) CD34+ hematopoietic stem and progenitor cells (HPSCs). Single element vectors containing modified boundaries and fragments of the E2, E9 and H53 elements are also included to determine if the modified (smaller sized elements) used to create the lead candidates still retain expression of the parental elements. WAS1.6 (the current WAS vector undergoing clinical trials) and the previously used y-retroviral vector are also included for comparison of expression.

[0246] As shown in Figure 22 the modified "slim" versions of E9, H53 and E2 retain expression of the larger parental elements. Additionally, one lead candidate vector, E9(slim)-H53(slim)-1,4,5(slim) of E2-HS 1pro-mCit-WRPE, has similar level of expression compare to the y-retroviral vector and a 1.8-fold higher expression than WAS1.6 in CB
CD34+ HSPC
differentiated megakaryocytes (Figure 22).

[0247] A similar result was seen in the CB CD34+ differentiated platelets as E9(slim)-H53(slim)-1,4,5(slim) of E2-HS1pro-mCit-WRPE expresses similar to the y-retroviral vector and 1.5 fold higher than WAS1.6 (see, e.g., Figure 23).

[0248] In view of these data, one lead candidate vector is E9(slim)-H53(slim)-1,4,5(slim) of E2-HS1pro-mCit-WPRE (WasVec) which:

[0249] 1) Expresses equal/higher than the y-retro in CB CD34+
megakaryocytes;

[0250] 2) Expresses 1.8 fold greater than WAS1.6;

[0251] 5.6kb w/ mCitrine in the open reading frame;

[0252] 6.4kb w/ WASp in the open reading frame; and

[0253] 3) Expresses 1.5 fold greater than WAS1.6 in "platelets".

[0254] In order to screen for the ideal codon optimized sequence of WASp to further improve expression, we immortalized T and B cell lines from WAS patient and used the cell lines to screen different codon optimized versions of the WASp open reading frame in WAS Vec ¨ (E9(slim)-HS3(slim)-1,4,5(slim) of E2-HS 1pro-WASp-WPRE)

[0255] The WAS B-cell and T-cell lines were transduced with different versions of WAS Vec, each encoding a different codon optimized version of the WASp open reading frame as follows:

[0256] cDNA (native cDNA sequence, unmodified as a control);

[0257] jCAT codon optimization;

[0258] GeneArt codon optimization;

[0259] IDT codon optimization; and

[0260] Benchling codon optimization.

[0261] As shown in Figures 24, and 25, jCAT codon optimization of WASP
in WAS Vec led to a 1.29 fold and 1.48 fold increase in expression in B cells (Figure 24) and T
cells (Figure 25) respectively compared to the non-codon optimized native cDNA. Figures 24, and 25 also show that GeneArt codon optimization led to a 1.13 fold and 1.20 fold increase in expression in B and T cells respectively compare to the native cDNA, while both IDT and Benchling codon optimizations led to a decrease in expression in B-cells and T-cells. None of the codon optimizations seem to significantly affect titer.
Additionally, CAT
and GeneArt may slightly increase titer (Figure 26).

[0262] In view of these observations, one lead candidate vector is E9(slim)-H53(slim)-E2(1,4,5 slim)-HS1pro-WASP(jCAT codon optimized)-WPRE, aka WAS Vec.
This vector is 6.4kb with WASp in the open reading frame

[0263] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

SEQUENCE LISTING
SEQ ID NO:1 HS1pro (minimal endogenous promoter of the WAS gene) TCAGCCTCAGGCTACCTAGGTGCTTTAGAAAGGAGGCCACCCAGGCCCATGACT
ACTCCTTGCCACAGGGAGCCCTGCACACAGATGTGCTAAGCTCTCGCTGCCAGCC
AGAGGGAGGAGGGTCTGAGCCAGTCAGAAGGAGATGGGCCCCAGAGAGTAAGA
AAGGGGGAGGAGGACCCAAGCTGATCCAAAAGGTGGGTCTAAGCAGTCAAGTG
GAGGAGGGTTCCAATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACGAGGAGGC
CAGGCCCACCAAGGGGCCCCTGGAGGACTTGTTTCCCTTGTCCCTTGTGGTTTTTT
GCATTTCCTGTTCCCTTGCTGCTCATTGCGGAAGTTCCTCTTCTTACCCTGCACCC
AGAGCCTCGCCAGAGAAGACAAGGGCAGAAAGCACC
SEQ ID NO:2 Enhancer element 2 (Slim) GACCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCC
CCTTCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCC
TCCCCAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCC
CTGCTGCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACT
TGGGGAGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGG
CGATCTGGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGC
CTGGCAGAAAGGCTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTA
ACCTCTCTAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTT
CCATCTGCTGGGTGTTGTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGC
ACAGTAGGTGGTCAAGAAACAGGAACACAATTTGTCATGACGACAATGATTCCT
TCATATGCTAGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAA
GCCCTTCCTCATTTCTGTTGCAATTCCTCAGCCAGAGGAAAGCCCCACCACCTCT
GTCCTCCAGAAAGCCCTCCTCTTCCCAGAAGGCTGGCACACAGGGCTGGGGGCC
AGGCTTGACTTCCCTGTGGAGGGGTCTGAGGTTGGGGGGATGACCTGCAGAGGT
AGGGGAATTCAGAGAACTTGTTTTGATAGAAACTGAAACTCCTCCTCTTGCTGA
CGCTGCACTTGTGGGCAATCTGCACCTGTTCTCCCCTCCCAGAGGCCAGCTGGA
AGGGGGTCCCCCACCATTCTACCTCTGTTCCCATCTCAAACAGGGCTCAGAAAT
CCTCAGAGAGCCTGGATTATCTTCCTATAGGGTCCTACCATTTGCTTTAGAGACA
TCTGAGAACTGCCTACTCCCCAGGTAGCTGCCTGCCTCCCAGAAGAGCCACAGC
AAGTTCCGCTAAGGGCAAAAAAGGAAGCTAGGTGTCTACAGGGAACCTAAAAA
CAAACCACACTAACGTGTGTACACACAGCTGCAGAGGGAGACAGGGACCTGGT
CTGCTCACAGCCCTTGCCACTCCCCCATGGCCCATGTAGAAACAACCATGTGGC
CTTCACTCTGCCCACAGAAGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTT
CTGCTGAGTCAAAGCCACATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGTCA
TGTTTCCAGGAGGAGGTTATCTGAGCATAACAGGGACAGGGTGGGCCACAGGA
TACCTCTGAGGCTCAGGTTCCCACCTCCACTCCACCCTCCAACTCTCTGCTCCAC
GTTCCTTCCACTATTCAACACATGGCAGGAAGTCAGCCTGTAGGGCTTCACACT
ACAGTCTAAGCTGACTGCCATCTGTCCCCATCCAGGCGAGGCTGGAAACGGGGG
CAGCAAAAAGCCCAGGGACAAAGTCCCCTTCCCAGGCACATGTATTATGCACTC
TGCACCAAGGAAACCTCCAGATAAGGTCTAGAGACCAAGGGCCATGTACTTCG
GGGGAGAGGCCAGAGGACTTCTGAGGTTTTACAGAGAAAAGCCAAAGGCAGCC
CAGTCAGGGGAAATGTGTAGCCATAGTGCCGATAAGGAAAGGCCTTCAACCTG
CCCTGGTCAGCTCTTCCTGTAAGTAGAGGCCCCTTACCGAAGGCCCCAGTGGGA
GGAAGGGTCGGGAGTA

SEQ ID NO: 3 Pt half of Core Sub-Element 1 of Enhancer Element 2 GACCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCC
CCTTCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCC
TCCCCAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCC
CTGCTGCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACT
TGGGGAGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGG
C
SEQ ID NO:4 2" half of Core Sub-Element 1 of Enhancer Element 2 GATCTGGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCC
TGGCAGAAAGGCTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAA
CCTCTCTAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCC
ATCTGCTGGGTGTTGTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGCAC
AGTAGGTGGTCAAGAAACAGGAACACAATTTGTCATGACGACAATGATTCCTTC
ATATGCTAGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAAGC
CCTTCCTCATTTCTGTTGCAATTCCTCAGCCAGAGGAAAGCCCCA
SEQ ID NO:5 Core Sub-Element 2 of Enhancer Element 2 CCACCTCTGTCCTCCAGAAAGCCCTCCTCTTCCCAGAAGGCTGGCACACAGGGC
TGGGGGCCAGGCTTGACTTCCCTGTGGAGGGGTCTGAGGTTGGGGGGATGACCT
GCAGAGGTAGGGGAATTCAGAGAACTTGTTTTGATAGAAACTGAAACTCCTCCT
CTTGCTGACGCTGCACTTGTGGGCAATCTGCACCTGTTCTCCCCTCCCAGAGGCC
AGCTGGAAGGGGGTCCCCCACCATTCTACCTCTGTTCCCATCTCAAACAGGGCT
CAGAAATCCTCA
SEQ ID NO:6 Core Sub-Element 3 of Enhancer Element 2 GAGAGCCTGGATTATCTTCCTATAGGGTCCTACCATTTGCTTTAGAGACATCTGA
GAACTGCCTACTCCCCAGGTAGCTGCCTGCCTCCCAGAAGAGCCACAGCAAGTT
CCGCTAAGGGCAAAAAAGGAAGCTAGGTGTCTACAGGGAACCTAAAAACAAAC
CACACTAACGTGTGTACACACAGCTGCAGAGGGAGACAGGGACCTGGTCTGCT
SEQ ID NO:7 Core-Sub Element 4 of Enhancer Element 2 CACAGCCCTTGCCACTCCCCCATGGCCCATGTAGAAACAACCATGTGGCCTTCA
CTCTGCCCACAGAAGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTTCTGCT
GAGTCAAAGCCACATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTT
CCAGGAGGAGGTTATCTGAGCATAACAGGGACAGGGTGGGCCACAGGATACCT
CTGAGGCTCAGGTTCCCACCTCCACTCCACC
SEQ ID NO: 8 Core-Sub Element 5 of Enhancer Element 2 CTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCAACACATGGCAGGAAGTCAG
CCTGTAGGGCTTCACACTACAGTCTAAGCTGACTGCCATCTGTCCCCATCCAGGC
GAGGCTGGAAACGGGGGCAGCAAAAAGCCCAGGGACAAAGTCCCCTTCCCAGG
CACATGTATTATGCACTCTGCACCAAGGAAACCTCCAGATAAGGTCTAGAGACC
AAGGGCCATGTACTTCGGGGGAGAGGCCAGAGGACTTCTGAGGTTTTACAGAG
AAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGTGTAGCCATAGTGCCGATAAG
GAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTAGAGGCCCCTTAC
CGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTA

SEO ID NO:9Enhancer element H53 (full) CTAAAAGCCACCAAAACCAAGACAGCAATGAAAGTAACCTCTGGTCTTCCT
AACTGCTCATTATACACTAATTATGATGCACTAGCATGCTAAGAGACACTTC
CACCAGCGCCATGACAGTGCCATGGCAACATCAAGAAGTCACCCCACATGG
TCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCCCACCCCTTTCCTG
GAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATTAAGAAATAACT
GTAAGTATCCTTAGTCCAGCAGCCCAAGCTGCTGCTCTGCCTATGGAGTAG
CCATTCTTTATTCCTTTACTTTCTTAATAAACTTGCTTTCACATTAAAAACAA
CAACAACAACACCAACAACCAGCAAATATATCAGAGGCTTAGCATCCCCTA
TTCAAGGGGACAGTTCTGGAGGCCGAGAAAGAAGTAGAAAGGTGGAGAAT
AAAAGACACAAAGGAGCAGAAGAATTTTATCTTGGCCTCCATGTGGCACCT
CACAGCTTGGGAACAACA
SEO ID NO:10 enhancer element H53 core CTAAAAGCCACCAAAACCAAGACAGCAATGAAAGTAACCTCTGGTCTTCCTAAC
TGCTCATTATACACTAATTATGATGCACTAGCATGCTAAGAGACACTTCCACCA
GCGCCATGACAGTGCCATGGCAACATCAAGAAGTCACCCCACATGGTCTAAAA
AGGGGAAGAACCTTCAGTTCCCGGAATTGCCCACCCCTTTCCTGGAAAACTCAT
GAATAATCCACCCCTTGTTTAGCATATAAT
SEO ID NO:11 Enhancer element E9 (full) GGGATCTGTGTGCAGATTTACCTCTACCTACACCTGGCTGGGGATCATAAAGAA
AATCAAGGGATGCGCTACCTCCATGAATCCAGGTTTCAGCAGA GCTAAGGGA GT
GAAATTTGGGGCCATTTTACTTGTCTCAAACCTTGTTATCTGAGAGAAAGCTAG
AGCTTCCTTCTTTCAGCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGGCTGGG
AAGATGAGCAATGCTTGTGAGTCACCATGATAGGAAGCAGAAGGGTCAGGAAG
TCCCTGGGAGCAAGGCTTAGGGTTAGGGTTAGGGAGAAAACACTTGGGCCTGG
AGGCTCGGGGCAGGCTTCCTAGAGGGGAGGGGTAGGAAGAGGCAGTGACAGGG
CCCACAGCAATGGAGAGGAGTTGGACTGCAGGGATGGGGTAGGGGGACAGACG
ACAAGGGACACTCAGAAGACTAATGTCTGGGAGTGGGAAAACAGTGTTTGCTC
AGCCAGGAAGCTGCATCCAGCTCTTTTATCATTTGTAGAAGACCAAGTACCCAG
GCCTGGGAGGAGCCCAGAGA
SEO ID NO:12 Enhancer element E9 core CAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCCTTCTTTCAGCCCCCAGAGA
CAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGAGCAATGCTTGTGAGTCAC
CATGATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGGAGCAAGGCTTAGGGTTA
GGGTTAGGGAGAAAACACTTGGGCCTGGAGGCTCGGGGCAGGCTTCCTAGAGG
GGAGGGGTA
SEO ID NO:13 WAS cDNA
atgagtgggggcccaatgggaggaaggcccgggggccgaggagcaccagcggacagcagaacataccctccaccctcct ccag gaccacgagaaccagcgactcatgagatgcaggacgaaaatgcttgacgctggccactgcagagttcagctgtacctgg cgctgcc ccctggagctgagcactggaccaaggagcattgtggggctgtgtgcttcgtgaaggataacccccagaagtcctacttc atccgccat acggccacaggctggtcggctgctctgggaacaggagctgtactcacagcttgtctactccacccccacccccacttcc acaccacg ctggagatgactgccaagcggggctgaactagcagacgaggacgaggcccaggccaccgggccctcgtgcaggagaaga taca aaaaaggaatcagaggcaaagtggagacagacgccagctacccccaccaccaacaccagccaatgaagagagaagagga gggc tcccacccctgcccctgcatccaggtggagaccaaggaggccctccagtgggtccgctctccctggggctggcgacagt ggacatc cagaaccctgacatcacgagttcacgataccgtgggctcccagcacctggacctagcccagctgataagaaacgctcag ggaagaa gaagatcagcaaagctgatattggtgcacccagtggattcaagcatgtcagccacgtggggtgggacccccagaatgga tttgacgt gaacaacctcgacccagatctgcggagtctgactccagggcaggaatcagcgaggcccagctcaccgacgccgagacct ctaaac ttatctacgacttcattgaggaccagggtgggctggaggctgtgcggcaggagatgaggcgccaggagccacttccgcc gccccca ccgccatctcgaggagggaaccagctcccccggccccctattgtggggggtaacaagggtcgactggtccactgccccc tgtacctt tggggattgccccacccccaccaacaccccggggacccccacccccaggccgagggggccctccaccaccaccccctcc agcta ctggacgactggaccactgccccctccaccccctggagctggtgggccacccatgccaccaccaccgccaccaccgcca ccgccg cccagctccgggaatggaccagcccctcccccactccctcctgctctggtgcctgccgggggcctggcccctggtgggg gtcgggg agcgcttaggatcaaatccggcagggaattcagctgaacaagacccctggggccccagagagctcagcgctgcagccac cacctc agagctcagagggactggtgggggccctgatgcacgtgatgcagaagagaagcagagccatccactcctccgacgaagg ggagg accaggctggcgatgaagatgaagatgatgaatgggatgactga SEQ ID NO:14 Codon optimized WAS
ATGAGCGGCGGCCCCATGGGCGGCCGCCCCGGCGGCCGCGGCGCCCCCGCCGTG
CAGCAGAACATCCCCAGCACCCTGCTGCAGGACCACGAGAACCAGCGCCTGTTC
GAGATGCTGGGCCGCAAGTGCCTGACCCTGGCCACCGCCGTGGTGCAGCTGTAC
CTGGCCCTGCCCCCCGGCGCCGAGCACTGGACCAAGGAGCACTGCGGCGCCGTG
TGCTTCGTGAAGGACAACCCCCAGAAGAGCTACTTCATCCGCCTGTACGGCCTGC
AGGCCGGCCGCCTGCTGTGGGAGCAGGAGCTGTACAGCCAGCTGGTGTACAGCA
CCCCCACCCCCTTCTTCCACACCTTCGCCGGCGACGACTGCCAGGCCGGCCTGAA
CTTCGCCGACGAGGACGAGGCCCAGGCCTTCCGCGCCCTGGTGCAGGAGAAGAT
CCAGAAGCGCAACCAGCGCCAGAGCGGCGACCGCCGCCAGCTGCCCCCCCCCCC
CACCCCCGCCAACGAGGAGCGCCGCGGCGGCCTGCCCCCCCTGCCCCTGCACCCC
GGCGGCGACCAGGGCGGCCCCCCCGTGGGCCCCCTGAGCCTGGGCCTGGCCACC
GTGGACATCCAGAACCCCGACATCACCAGCAGCCGCTACCGCGGCCTGCCCGCC
CCCGGCCCCAGCCCCGCCGACAAGAAGCGCAGCGGCAAGAAGAAGATCAGCAA
GGCCGACATCGGCGCCCCCAGCGGCTTCAAGCACGTGAGCCACGTGGGCTGGGA
CCCCCAGAACGGCTTCGACGTGAACAACCTGGACCCCGACCTGCGCAGCCTGTTC
AGCCGCGCCGGCATCAGCGAGGCCCAGCTGACCGACGCCGAGACCAGCAAGCTG
ATCTACGACTTCATCGAGGACCAGGGCGGCCTGGAGGCCGTGCGCCAGGAGATG
CGCCGCCAGGAGCCCCTGCCCCCCCCCCCCCCCCCCAGCCGCGGCGGCAACCAG
CTGCCCCGCCCCCCCATCGTGGGCGGCAACAAGGGCCGCAGCGGCCCCCTGCCC
CCCGTGCCCCTGGGCATCGCCCCCCCCCCCCCCACCCCCCGCGGCCCCCCCCCCC
CCGGCCGCGGCGGCCCCCCCCCCCCCCCCCCCCCCGCCACCGGCCGCAGCGGCCC
CCTGCCCCCCCCCCCCCCCGGCGCCGGCGGCCCCCCCATGCCCCCCCCCCCCCCC
CCCCCCCCCCCCCCCCCCAGCAGCGGCAACGGCCCCGCCCCCCCCCCCCTGCCCC
CCGCCCTGGTGCCCGCCGGCGGCCTGGCCCCCGGCGGCGGCCGCGGCGCCCTGCT
GGACCAGATCCGCCAGGGCATCCAGCTGAACAAGACCCCCGGCGCCCCCGAGAG
CAGCGCCCTGCAGCCCCCCCCCCAGAGCAGCGAGGGCCTGGTGGGCGCCCTGAT
GCACGTGATGCAGAAGCGCAGCCGCGCCATCCACAGCAGCGACGAGGGCGAGG
ACCAGGCCGGCGACGAGGACGAGGACGACGAGTGGGACGACTAA
SEQ ID NO:15 E2(all slim)-HS1pro-mCit-WPRE
Sequence from CMV (red arrow in Figure 18) going clockwise:
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG
ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
GGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGG
CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACG
GTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC

TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGG
CAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTC
CACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT
CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTA
CGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGT
TAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTA
AGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTG
TGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCT
AGCagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctga agc gcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattagactagcggaggctagaaggagagagatg ggt gcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaa atataa attaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggc tgtagacaa atactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctct attgtgtgcat caaaggatagagataaaagacaccaaggaagattagacaagatagaggaagagcaaaacaaaagtaagaccaccgcaca gca agcggccgctgatatcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagta aaaattga accattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagattga cca gggacttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctgg tatagtg cagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgagcaactcacagtctggggcatcaagcagct ccaggc aagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggataggggagctctggaaaactcatttgcac cactgct gtgccaggaatgctagaggagtaataaatctctggaacagataggaatcacacgacctggatggagtgggacagagaaa ttaaca attacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaatt agataaatgg gcaagtagtggaattggataacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcaggtag gataagaat agtattgctgtactactatagtgaatagagttaggcagggatattcaccattatcgatcagacccacctcccaaccccg aggggaccc gacaggcccgaaggaatagaagaagaaggtggagagagagacag agacagatccattcgattagtgaacggatctcgacggtat cggttaactataaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacat acaaac taaagaattacaaaaacaaattacaaaaattcaaaatatatcgatcacgagactagcctcgagGACCTGAAGGGTAAG

GGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCTTCACCAATGTAA
ACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCCTCCCCAGCGGCATTT
CCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCTGCTGCCTTTC
TTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACTTGGGGAGCTATTTCT
GTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGGCGATCTGGGGGAAA
ACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCCTGGCAGAAAGGCT
CTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAACCTCTCTAAGCTGA
AATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCCATCTGCTGGGTGTT
GTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGCACAGTAGGTGGTCAA
GAAACAGGAACACAATTTGTCATGACGACAATGATTCCTTCATATGCTAGTTAC
CCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAAGCCCTTCCTCATTTCT
GTTGCAATTCCTCAGCCAGAGGAAAGCCCCACCACCTCTGTCCTCCAGAAAGCC
CTCCTCTTCCCAGAAGGCTGGCACACAGGGCTGGGGGCCAGGCTTGACTTCCCT
GTGGAGGGGTCTGAGGTTGGGGGGATGACCTGCAGAGGTAGGGGAATTCAGAG
AACTTGTTTTGATAGAAACTGAAACTCCTCCTCTTGCTGACGCTGCACTTGTGGG
CAATCTGCACCTGTTCTCCCCTCCCAGAGGCCAGCTGGAAGGGGGTCCCCCACC
ATTCTACCTCTGTTCCCATCTCAAACAGGGCTCAGAAATCCTCAGAGAGCCTGG
ATTATCTTCCTATAGGGTCCTACCATTTGCTTTAGAGACATCTGAGAACTGCCTA
CTCCCCAGGTAGCTGCCTGCCTCCCAGAAGAGCCACAGCAAGTTCCGCTAAGGG
CAAAAAAGGAAGCTAGGTGTCTACAGGGAACCTAAAAACAAACCACACTAACG
TGTGTACACACAGCTGCAGAGGGAGACAGGGACCTGGTCTGCTCACAGCCCTTG
CCACTCCCCCATGGCCCATGTAGAAACAACCATGTGGCCTTCACTCTGCCCACA
GAAGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTTCTGCTGAGTCAAAGCC
ACATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTTCCAGGAGGAGG
TTATCTGAGCATAACAGGGACAGGGTGGGCCACAGGATACCTCTGAGGCTCAG
GTTCCCACCTCCACTCCACCCTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCA

ACACATGGCAGGAAGTCAGCCTGTAGGGCTTCACACTACAGTCTAAGCTGACTG
CCATCTGTCCCCATCCAGGCGAGGCTGGAAACGGGGGCAGCAAAAAGCCCAGG
GACAAAGTCCCCTTCCCAGGCACATGTATTATGCACTCTGCACCAAGGAAACCT
CCAGATAAGGTCTAGAGACCAAGGGCCATGTACTTCGGGGGAGAGGCCAGAGG
ACTTCTGAGGTTTTACAGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGT
GTAGCCATAGTGCCGATAAGGAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCC
TGTAAGTAGAGGCCCCTTACCGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTA
ctcgagTCAGCCTCAGGCTACCTAGGTGCTTTAGAAAGGAGGCCACCCAGGCCCAT
GACTACTCCTTGCCACAGGGAGCCCTGCACACAGATGTGCTAAGCTCTCGCTGC
CAGCCAGAGGGAGGAGGGTCTGAGCCAGTCAGAAGGAGATGGGCCCCAGAGA
GTAAGAAAGGGGGAGGAGGACCCAAGCTGATCCAAAAGGTGGGTCTAAGCAGT
CAAGTGGAGGAGGGTTCCAATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACG
AGGAGGCCAGGCCCACCAAGGGGCCCCTGGAGGACTTGTTTCCCTTGTCCCTTG
TGGTTTTTTGCATTTCCTGTTCCCTTGCTGCTCATTGCGGAAGTTCCTCTTCTTAC
CCTGCACCCAGAGCCTCGCCAGAGAAGACAAGGGCAGAAAGCACCATGgtgagcaa gggcgaggagctgacaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgt ccgg cgagggcgagggcgatgccacctacggcaagctgaccctgaagttc atctgcaccaccggcaagctgcccgtgccctggccc ac cctcgtgaccaccacggctacggcctgatgtgcttcgcccgctaccccgaccacatgaagcagcacgacttatcaagtc cgccatg cccgaaggctacgtccaggagcgcaccatcacttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcga gggc gacaccctggtgaaccgcatcgagctgaagggcatcgacttcaagg aggacggcaacatcctggggcacaagctggagtacaac tacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaaca tcgagg acggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaacca ctacc tgagctaccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgc cgggat cactctcggcatggacgagctgtacaagtgactgcaggaattcgagcatcttaccgccatttattcccatatttgactg atacttgatttg ggtatacatttaaatgttaataaaacaaaatggtggggcaatcatttacattatagggatatgtaattactagttcagg tgtattgccacaa gacaaacatgttaagaaactacccgttatttacgctctgacctgttaatcaacctctggattacaaaatagtgaaagat tgactgatattc ttaactatgagctccattacgctgtgtggatatgctgattaatgcctctgtatcatgctattgatcccgtacggattcg attctcctcctt gtataaatcctggagctgtctattatgaggagagtggcccgagtccgtcaacgtggcgtggtgtgctctgtgatgctga cgcaacc cccactggctggggcattgccaccacctgtcaactcctuctgggactacgattccccctcccgatcgccacggcagaac tcatcgc cgcctgccttgcccgctgctggacaggggctaggagctgggcactgataattccgtggtgagtcggggaagggcctgct gccgg ctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaat tcgagctcgg taccataagaccaatgacttacaaggcagctgtagatcttagccactattaaaagaaaaggggggactggaagggctaa ttcactcc caacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggct aactagggaac ctactgcttaagcctcaataaagcttgccttgagtgcttCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTC
TGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCagta gtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgatatt gcagcttataatgg ttacaaataaagcaatagcatcacaaatttcacaaataaagcattatttcactgcattctagagtggtagtccaaactc atcaatgtatctt atcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagaccgcccattctccgc cccatggct gactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttt tggaggcctag ggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgattacaacgtcgtgactgggaaa accctggc gttacccaacttaatcgccagcagcacatccccattcgccagctggcgtaatagcgaagaggcccgcaccgatcgccat cccaa cagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgca gcgtg accgctacacttgccagcgccctagcgcccgctcattcgattcaccatcattctcgccacgttcgccggctaccccgtc aagctc taaatcgggggctccattagggaccgatttagtgattacggcacctcgaccccaaaaaacttgattagggtgatggaca cgtagtg ggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaac tggaacaacact caaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatt taacaaaaatttaac gcgaatataacaaaatattaacgcttacaatttaggtggcactatcggggaaatgtgcgcggaacccctatttgatatt atctaaatac attcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacctagatcaagagacaggatg aggatcgatc gcatgattgaacaagatggattgcacgcaggactccggccgcagggtggagaggctattcggctatgactgggcacaac agac a atcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccg gtgccctga atgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgaccagcgcagctgtgctcgacgagtcac tgaa gcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccagctcctgccgagaaagt atccatc atggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcg agcgagc acgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactg ttcgcca ggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtgga aaatggc cgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgata ttgctgaaga gcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctat cgccttcttga cgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacac cgcatcaggtggca cttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaccaa aatcccttaacgtg agttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaat ctgctgcttgcaa acaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggct tcagcagag cgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacata cctcgctctgc taatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccgga taaggcgca gcggtcgggctgaacggggggttcgtgcac acagcccagcttggagcgaacgacctac accgaactgagatacctacagcgtga gctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagag cgc acgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgat ttttgtgatgc tcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttg ctcacatgtt ctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga acgaccgagc gcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcatta atgca gctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggc accccag gctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatg accatgattacg ccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttggccattgcatacgttgtatccata tcataatat gtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaatt acggggtcattagttc atagcccatatatgg SEQ ID NO:16 E9(slim)-HS3(slim)-E2(all slim)-HS1pro-mCit-WPRE (added the E9(slim) and H53(slim) elements) Sequence from CMV (red arrow) going clockwise in Figure 19:
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA
CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG
ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG
TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA
ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGG
CAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT
ACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCC
CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA
TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG
GAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCA
GATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA
TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGG
TAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcc cgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggc aagag gcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgt cagtat taagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacat atagtatg ggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactggga cagctaca accatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagg atagagataaaa gacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatc ttcaga cctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggag tagcaccca ccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagc agcagga agcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaaca atttgctga gggctattgaggcgcaacagcatctgttgcaactcac agtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagat acctaaaggatcaacagctcctggggataggggagctctggaaaactcatttgcaccactgctgtgccaggaatgctag aggagta ataaatctctggaacagataggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaata cactccttaa ttgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtagtggaattgg ataacataa caaattggctgtggtatataaaattattcataatgatagtaggaggcaggtaggataagaatagtattgctgtactact atagtgaataga gttaggcaggg atattcaccattatcgatcag acccacctcccaaccccg aggggacccg acaggcccg aagg aatag aagaag a aggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaactataaaagaaaag gggggatt ggggggtacagtgcagggg aaagaatagtag acataatagcaacagacatacaaactaaag aattacaaaaacaaattacaaaaatt caaaatatatcgatcacgagactagcctcgagCAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCC
TTCTTTCAGCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGA
GCAATGCTTGTGAGTCACCATGATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGG
AGCAAGGCTTAGGGTTAGGGTTAGGGAGAAAACACTTGGGCCTGGAGGCTCGGG
GCAGGCTTCCTAGAGGGGAGGGGTACTAAAAGCCACCAAAACCAAGACAGCAAT
GAAAGTAACCTCTGGTCTTCCTAACTGCTCATTATACACTAATTATGATGCACTA
GCATGCTAAGAGACACTTCCACCAGCGCCATGACAGTGCCATGGCAACATCAAG
AAGTCACCCCACATGGTCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCC
CACCCCTTTCCTGGAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATGA
CCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCT
TCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCCTCCC
CAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCT
GCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACTTGGGG
AGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGGCGATCT
GGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCCTGGCA
GAAAGGCTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAACCTCTC
TAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCCATCTGC
TGGGTGTTGTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGCACAGTAGG
TGGTCAAGAAACAGGAACACAATTTGTCATGACGACAATGATTCCTTCATATGCT
AGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAAGCCCTTCCTC
ATTTCTGTTGCAATTCCTCAGCCAGAGGAAAGCCCCACCACCTCTGTCCTCCAGA
AAGCCCTCCTCTTCCCAGAAGGCTGGCACACAGGGCTGGGGGCCAGGCTTGACTT
CCCTGTGGAGGGGTCTGAGGTTGGGGGGATGACCTGCAGAGGTAGGGGAATTCA
GAGAACTTGTTTTGATAGAAACTGAAACTCCTCCTCTTGCTGACGCTGCACTTGT
GGGCAATCTGCACCTGTTCTCCCCTCCCAGAGGCCAGCTGGAAGGGGGTCCCCCA
CCATTCTACCTCTGTTCCCATCTCAAACAGGGCTCAGAAATCCTCAGAGAGCCTG
GATTATCTTCCTATAGGGTCCTACCATTTGCTTTAGAGACATCTGAGAACTGCCTA
CTCCCCAGGTAGCTGCCTGCCTCCCAGAAGAGCCACAGCAAGTTCCGCTAAGGG
CAAAAAAGGAAGCTAGGTGTCTACAGGGAACCTAAAAACAAACCACACTAACGT
GTGTACACACAGCTGCAGAGGGAGACAGGGACCTGGTCTGCTCACAGCCCTTGC
CACTCCCCCATGGCCCATGTAGAAACAACCATGTGGCCTTCACTCTGCCCACAGA
AGCTAGCACCAGGACCCTGGTCAGGGTTAGAGGTTTCTGCTGAGTCAAAGCCAC
ATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTTCCAGGAGGAGGTT
ATCTGAGCATAACAGGGACAGGGTGGGCCACAGGATACCTCTGAGGCTCAGGTT
CCCACCTCCACTCCACCCTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCAACA
CATGGCAGGAAGTCAGCCTGTAGGGCTTCACACTACAGTCTAAGCTGACTGCCAT
CTGTCCCCATCCAGGCGAGGCTGGAAACGGGGGCAGCAAAAAGCCCAGGGACA
AAGTCCCCTTCCCAGGCACATGTATTATGCACTCTGCACCAAGGAAACCTCCAGA
TAAGGTCTAGAGACCAAGGGCCATGTACTTCGGGGGAGAGGCCAGAGGACTTCT
GAGGTTTTACAGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGTGTAGCCA
TAGTGCCGATAAGGAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTA
GAGGCCCCTTACCGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTActcgagTCAGC
CTCAGGCTACCTAGGTGCTTTAGAAAGGAGGCCACCCAGGCCCATGACTACTCCT
TGCCACAGGGAGCCCTGCACACAGATGTGCTAAGCTCTCGCTGCCAGCCAGAGG
GAGGAGGGTCTGAGCCAGTCAGAAGGAGATGGGCCCCAGAGAGTAAGAAAGGG

GGAGGAGGACCCAAGCTGATCCAAAAGGTGGGTCTAAGCAGTCAAGTGGAGGA
GGGTTCCAATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACGAGGAGGCCAGGC
CCACCAAGGGGCCCCTGGAGGACTTGTTTCCCTTGTCCCTTGTGGTTTTTTGCATT
TCCTGTTCCCTTGCTGCTCATTGCGGAAGTTCCTCTTCTTACCCTGCACCCAGAGC
CTCGCCAGAGAAGACAAGGGCAGAAAGCACCATGgtgagcaagggcgaggagctgttcaccggg gtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatg ccacct acggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccacggc tacggcc tgatgtgcttcgcccgctaccccgaccacatgaagcagcacgacttatcaagtccgccatgcccgaaggctacgtccag gagcgca ccatcacttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatc gagctg aagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctata tcatggc cgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgac cactac cagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagca aagacc ccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgta caagtga ctgcaggaattcgagcatcttaccgccatttattcccatatttgactgatacttgatagggtatacatttaaatgttaa taaaacaaaatggt ggggcaatcatttacattatagggatatgtaattactagttcaggtgtattgccacaagacaaacatgttaagaaacta cccgttatttacg ctctgacctgttaatcaacctctggattacaaaatagtgaaagattgactgatattcttaactatgagctccattacgc tgtgtggatatgct gctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctccttgtataaatcctggttgctgt ctctttatgaggagttgt ggcccgagtccgtcaacgtggcgtggtgtgctctgtgatgctgacgcaacccccactggctggggcattgccaccacct gtcaactc catctgggactacgctaccccctcccgatcgccacggcagaactcatcgccgcctgccttgcccgctgctggacagggg ctaggtt gctgggcactgataattccgtggtgagtcggggaagggcctgctgccggctctgcggcctcaccgcgtcttcgccacgc cctcaga cgagtcggatctccctagggccgcctccccgcctggaattcgagctcggtaccataagaccaatgacttacaaggcagc tgtagatct tagccactattaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctattgatgtact gggtctctc tggttagaccagatctgagcctgggagctctctggctaactagggaacctactgcttaagcctcaataaagcttgcctt gagtgcttCA
AGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC
TTTTAGTCAGTGTGGAAAATCTCTAGCagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaa atgaatatcagagagtgagaggaacttgatattgcagcttataatggttacaaataaagcaatagcatcacaaatttca caaataaagcat ttattcactgcattctagagtggtagtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgccccta actccgcccatccc gcccctaactccgcccagaccgcccattctccgccccatggctgactaattatatatttatgcagaggccgaggccgcc tcggcctct gagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtatta cgcgcgctcac tggccgtcgattacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccagcagcacatccccattcgc cagctggc gtaatagcgaagaggcccgcaccgatcgccatcccaacagagcgcagcctgaatggcgaatgggacgcgccctgtagcg gcgc attaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcattcgatt cacccttc catctcgccacgttcgccggctaccccgtcaagctctaaatcgggggctccattagggaccgatttagtgattacggca cctcgacc ccaaaaaacttgattagggtgatggacacgtagtgggccatcgccctgatagacggtattcgccattgacgaggagtcc acgttatt aatagtggactcttgaccaaactggaacaacactcaaccctatctcggtctattcattgatttataagggattagccga tttcggcctattg gttaaaaaatgagctgatttaacaaaaatttaacgcgaatataacaaaatattaacgcttacaatttaggtggcactat cggggaaatgtg cgcggaacccctatttgatatttactaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgctt caataatagcacc tagatcaagagacaggatgaggatcgatcgcatgattgaacaagatggattgcacgcaggactccggccgcttgggtgg agaggct attcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgaccggctgtcagcgcaggggcgcccgg actattg tcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcga ccagc gcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgt catctcac cagctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccatt cgaccacca agcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcagtcgatcaggatgatctggacgaagagcatc aggggc tcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgc ctgcttg ccgaatatcatggtggaaaatggccgcttactggattcatcgactgtggccggctgggtgtggcggaccgctatcagga catagcgtt ggctacccgtgatattgctgaagagcaggcggcgaatgggctgaccgatcctcgtgattacggtatcgccgctcccgat tcgcagc gcatcgccactatcgccacttgacgagttcactgaattattaacgcttacaatacctgatgcggtattactccttacgc atctgtgcggta tacacaccgcatcaggtggcactatcggggaaatgtgcgcggaacccctatttgatattatctaaatacattcaaatat gtatccgctca tgaccaaaatcccttaacgtgagattcgaccactgagcgtcagaccccgtagaaaagatcaaaggatcacttgagatca ttattctgc gcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggatgatgccggatcaagagctaccaactatta ccgaagg taactggcttcagcagagcgcagataccaaatactgacactagtgtagccgtagttaggccaccacttcaagaactctg tagcaccgc ctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggaggactca agacgatagtt accggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaa ctgag atacctac agcgtg agctatg ag aaagcgccacgcttcccg aaggg agaaaggcgg ac aggtatccggtaagcggc agggtcgg a acaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgac ttgagcgtc gatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggcctt ttgctggccttt tgctcacatgttattcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctc gccgcagccgaa cgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgccc aatacgcaaaccgcctctccccgcgcgttggccgattc attaatgcagctggcacgac aggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggc accccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataac aatttcac ac aggaaac agctatg acc at gattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttggccattgcatacgttgt atccatatca taatatgtacatttatattggctcatgtccaacattaccgccatgttgac attgattattgactagttattaatagtaatcaattacggggtcatt agttcatagcccatatatgg SEQ ID NO:17 E9(slim)-HS3(slim)-E2(1,4,5 slim)-HS1pro-mCit-WPRE
(Deleted Core Sub-Element 2 of Element 2 and Core Sub-Element 3 of Element 2) Sequence from CMV (red arrow) going clockwise in Figure 20 AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA
CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG
ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG
TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA
ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGG
CAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT
ACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCC
CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA
TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG
GAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCA
GATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA
TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGG
TAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcc cgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggc aagag gcg aggggcggcg actggtgagtacgcc aaaaattttg actagcgg aggctagaaggag agagatgggtgcgag agcgtcagtat taagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacat atagtatg ggc aagc agggagctag aacgattcgc agttaatcctggcctgttag aaac atc ag aaggctgtag ac aaatactggg ac agctac a accatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagg atagagataaaa gac accaaggaagctttagac aag atag agg aagagc aaaac aaaagtaag acc accgc ac agc aagcggccgctg atcttc ag a cctggaggagg ag atatgaggg ac aattggagaagtgaattatataaatataaagtagtaaaaattgaacc attaggagtagc accc a cc aaggc aaag ag aag agtggtgc agag agaaaaaagagc agtgggaatagg agctttgttccttgggttcttggg agc agc agga agcactatgggcgcagcgtc aatgacgctgacggtacaggcc agacaattattgtctggtatagtgc agcagcagaacaatttgctga gggctattgaggcgcaacagcatctgttgcaactcac agtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagat acctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgc tagttggagta ataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaat acactccttaa ttgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattg gtttaacataa caaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtact ttctatagtgaataga gttaggc aggg atattc acc attatcgtttcag accc acctccc aaccccg aggggacccg ac aggcccg aagg aatag aagaag a aggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaagaaaa ggggggatt ggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaatta caaaaatt caaaattttatcgatcacgag actagcctcgagCAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCC
TTCTTTCAGCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGA
GCAATGCTTGTGAGTCACCATGATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGG
AGCAAGGCTTAGGGTTAGGGTTAGGGAGAAAACACTTGGGCCTGGAGGCTCGGG

GCAGGCTTCCTAGAGGGGAGGGGTACTAAAAGCCACCAAAACCAAGACAGCAAT
GAAAGTAACCTCTGGTCTTCCTAACTGCTCATTATACACTAATTATGATGCACTA
GCATGCTAAGAGACACTTCCACCAGCGCCATGACAGTGCCATGGCAACATCAAG
AAGTCACCCCACATGGTCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCC
CACCCCTTTCCTGGAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATGA
CCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCT
TCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCCTCCC
CAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCT
GCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACTTGGGG
AGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGGCGATCT
GGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCCTGGCA
GAAAGGCTCTGCTGCCTCCTAGCTGTGTAGCAATGGATAAGGCCTTTAACCTCTC
TAAGCTGAAATGTCCTCATGCATAAGGTGGAGACAGTAACTCCTCTTCCATCTGC
TGGGTGTTGTCAAAACTACACAGGAGGTTTGCCAAGCACCTAAGGCACAGTAGG
TGGTCAAGAAACAGGAACACAATTTGTCATGACGACAATGATTCCTTCATATGCT
AGTTACCCACGAGTCAGCCAGAATGTTGCCATAACCACTTATGAAGCCCTTCCTC
ATTTCTGTTGCAATTCCTCAGCCAGAGGAAAGCCCCACACAGCCCTTGCCACTCC
CCCATGGCCCATGTAGAAACAACCATGTGGCCTTCACTCTGCCCACAGAAGCTAG
CACCAGGACCCTGGTCAGGGTTAGAGGTTTCTGCTGAGTCAAAGCCACATGGAG
GGAGGGAGCAAGGGAGAGATGCAGAGTCATGTTTCCAGGAGGAGGTTATCTGAG
CATAACAGGGACAGGGTGGGCCACAGGATACCTCTGAGGCTCAGGTTCCCACCT
CCACTCCACCCTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCAACACATGGCA
GGAAGTCAGCCTGTAGGGCTTCACACTACAGTCTAAGCTGACTGCCATCTGTCCC
CATCCAGGCGAGGCTGGAAACGGGGGCAGCAAAAAGCCCAGGGACAAAGTCCC
CTTCCCAGGCACATGTATTATGCACTCTGCACCAAGGAAACCTCCAGATAAGGTC
TAGAGACCAAGGGCCATGTACTTCGGGGGAGAGGCCAGAGGACTTCTGAGGTTT
TACAGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGTGTAGCCATAGTGCC
GATAAGGAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTAGAGGCCC
CTTACCGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTActcgagTCAGCCTCAGGC
TACCTAGGTGCTTTAGAAAGGAGGCCACCCAGGCCCATGACTACTCCTTGCCACA
GGGAGCCCTGCACACAGATGTGCTAAGCTCTCGCTGCCAGCCAGAGGGAGGAGG
GTCTGAGCCAGTCAGAAGGAGATGGGCCCCAGAGAGTAAGAAAGGGGGAGGAG
GACCCAAGCTGATCCAAAAGGTGGGTCTAAGCAGTCAAGTGGAGGAGGGTTCCA
ATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACGAGGAGGCCAGGCCCACCAAG
GGGCCCCTGGAGGACTTGTTTCCCTTGTCCCTTGTGGTTTTTTGCATTTCCTGTTCC
CTTGCTGCTCATTGCGGAAGTTCCTCTTCTTACCCTGCACCCAGAGCCTCGCCAGA
GAAGACAAGGGCAGAAAGCACCATGgtgagcaagggcgaggagctgttcaccggggtggtgcccatcctg gtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagc tgacc ctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcggctacggcctgatgt gcttcgccc gctaccccgaccacatgaagcagcacgacttatcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcact tcaagg acgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcat cgactt caaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaag cagaag aacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcaga acaccc ccatcggcgacggccccgtgctgctgcccgacaaccactacctgagctaccagtccgccctgagcaaagaccccaacga gaagcg cgatcacatggtcctgctggagttcgtgaccgccgccgggatc actctcggcatggacgagctgtacaagtgactgcaggaattcga gcatcttaccgccatttattcccatatttgactgatacttgatagggtatacatttaaatgttaataaaacaaaatggt ggggcaatcattta cattatagggatatgtaattactagttcaggtgtattgccacaagacaaacatgttaagaaactacccgttatttacgc tctgacctgttaat caacctctggattacaaaatagtgaaagattgactgatattcttaactatgagctccattacgctgtgtggatatgctg attaatgcctctg tatcatgctattgatcccgtacggattcgattctcctccagtataaatcctggagctgtctattatgaggagagtggcc cgagtccgtc aacgtggcgtggtgtgctctgtgatgctgacgcaacccccactggctggggcattgccaccacctgtcaactcattctg ggactacg ctttccccctcccgatcgccacggcagaactcatcgccgcctgccttgcccgctgctggacaggggctaggttgctggg cactgataa accgtggtgagtcggggaagggcctgctgccggctctgcggcctcaccgcgtcttcgccacgccctcagacgagtcgga tctccct ttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagcc actttttaaaag aaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggtt agaccagatct gagcctggg agctctctggctaactaggg aacctactgcttaagcctcaataaagcttgccttg agtgcttCAAGTAGTGT GT
GCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGT
GTGGAAAATCTCTAGCagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagag agtg agaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattttt ttcactgcattctagt tgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcc cctaactccgccc agttccgc cc attctccgc ccc atggctgactaattttttttatttatgc ag aggccg aggccgcctcggcctctg agctattcc agaagta gtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccg tcgttttacaa cgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccattcgccagctggcgtaatag cgaagagg cccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgc ggcgggt gtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttc tcgccacgttcg ccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaa aaaacttgatta gggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaat agtggactcttgtt cc aaactgg aac aac actcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctg atttaac aaaaatttaacgcg aattttaacaaaatattaacgcttac aatttaggtggcacttttcgggg aaatgtgcgcggaacccctattt gtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacct agatcaagagacag gatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcgg ctatgactgg gcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaaga ccgacctgt ccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgt gctcgac gttgtc actgaagcggg aaggg actggctgctattgggcgaagtgccggggc agg atctcctgtc atctc accttgctcctgccgag a aagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccacc aagcg aaac atcgc at cgagcgagcacgtactcgg atgg aagccggtcttgtcgatc agg atg atctgg acg aagagc atc aggggctcgcgccagccgaa ctgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaata tcatggtg gaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggcta cccgtgatatt gctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcg ccttctatcg ccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggt atttcacaccgcatcag gtggcacttttcggggaaatgtgc gcgg aacccctatttgtttatttttctaaatac attc aaatatgtatccgctc atgacc aaaatccctta acgtgagttttcgttcc actgagcgtc agaccccgtag aaaag atc aaagg atcttcttg ag atcctttttttctgcgcgtaatctgctgctt gcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaact ggcttcagca gagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctac atacctcgctct gctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccg gataaggcgc agcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctaca gcgtga gctatgagaaagcgccacgcttcccgaagggagaaaggcggac aggtatccggtaagcggc agggtcgg aac aggag agcgc a cgagggagcttccaggggg aaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctc gtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgct cacatgttcttt cctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacga ccgagcgcag cgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgc agctggc acgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccc aggctttac a ctttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgat tacgccaagcgc gcaattaaccctcactaaagggaacaaaagctggagctgcaagcttggccattgcatacgttgtatccatatcataata tgtacatttatat tggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcatta gttcatagcccatata tgg SEQ ID NO:18 E9(slim)-HS3(slim)-E2(1st half of 1 and 5 slim)-HS1pro-mCit-WPRE (Deleted 2nd half of Core Sub-Element 1 of Element 2 and Core-Sub Element of Element 2) Sequence from CMV (red arrow) going clockwise in Figure 21 AGTTCCGC GTTACATAACTTAC GGTAAAT GGCCC GCCTGGCTGACC GCCCAAC GA
CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGG
ACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG
TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAA
ATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGG
CAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGT
ACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCC
CATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA
TGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG
GAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCA
GATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAA
TAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGG
TAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcc cgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggc aagag gcgaggggcggcgactggtgagtacgccaaaaattagactagcggaggctagaaggagagagatgggtgcgagagcgtc agtat taagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacat atagtatg ggc aagc agggagctag aacgattcgc agttaatcctggcctgttag aaac atc ag aaggctgtag ac aaatactggg ac agctac a accatccatcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaagga tagagataaaa gacaccaaggaagattagacaagatagaggaagagcaaaacaaaagtaagacc accgc ac agc aagcggccgctg atcttc ag a cctggaggagg ag atatgaggg ac aattggagaagtgaattatataaatataaagtagtaaaaattgaacc attaggagtagc accc a cc aaggc aaag ag aag agtggtgc agag agaaaaaagagc agtgggaatagg agctttgttccttgggttcttggg agc agc agga agcactatgggcgcagcgtc aatgacgctgacggtacaggccagacaattattgtctggtatagtgc agcagcagaacaatttgctga gggctattgaggcgcaacagcatctgttgcaactcac agtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagat acctaaaggatcaacagctcctggggataggggagctctggaaaactcatttgcaccactgctgtgccaggaatgctag aggagta ataaatctctggaacagataggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaata cactccttaa ttgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtagtggaattgg ataacataa caaattggctgtggtatataaaattattcataatgatagtaggaggcaggtaggataagaatagtattgctgtactact atagtgaataga gttaggc aggg atattc acc attatcgatcag accc acctccc aaccccg aggggacccg ac aggcccg aagg aatag aagaag a aggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaactataaaagaaaag gggggatt ggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaatta caaaaatt caaaattttatcgatcacgag actagcctcgagCAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCC
TTCTTTCAGCCCCCAGAGACAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGA
GCAATGCTTGTGAGTCACCATGATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGG
AGCAAGGCTTAGGGTTAGGGTTAGGGAGAAAACACTTGGGCCTGGAGGCTCGGG
GCAGGCTTCCTAGAGGGGAGGGGTACTAAAAGCCACCAAAACCAAGACAGCAAT
GAAAGTAACCTCTGGTCTTCCTAACTGCTCATTATACACTAATTATGATGCACTA
GCATGCTAAGAGACACTTCCACCAGCGCCATGACAGTGCCATGGCAACATCAAG
AAGTCACCCCACATGGTCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCC
CACCCCTTTCCTGGAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATGA
CCTGAAGGGTAAGGGGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCT
TCACCAATGTAAACAAGGATGTGGGTTCTGCGGCCACACTCTCCCCCGCCCTCCC
CAGCGGCATTTCCAGCAAGTCACATGTCCTGCGCACAGGCTGGGGGCCCCCTGCT
GCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGCACAGTGAGCTACTTGGGG
AGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCTGTTGGCCTCCA
ACTCTCTGCTCCACGTTCCTTCCACTATTCAACACATGGCAGGAAGTCAGCCTGT
AGGGCTTCACACTACAGTCTAAGCTGACTGCCATCTGTCCCCATCCAGGCGAGGC
TGGAAACGGGGGCAGCAAAAAGCCCAGGGACAAAGTCCCCTTCCCAGGCACATG
TATTATGCACTCTGCACCAAGGAAACCTCCAGATAAGGTCTAGAGACCAAGGGC
CATGTACTTCGGGGGAGAGGCCAGAGGACTTCTGAGGTTTTACAGAGAAAAGCC
AAAGGCAGCCCAGTCAGGGGAAATGTGTAGCCATAGTGCCGATAAGGAAAGGCC
TTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTAGAGGCCCCTTACCGAAGGCCC
CAGTGGGAGGAAGGGTCGGGAGTActcgagTCAGCCTCAGGCTACCTAGGTGCTTT

-6 g-f oo-coounuuReanuofilofiofiovuif of ofiommooiefefiloilovffeReolauReufeif0000u fuoif of-0 fiouoolif oimfaif ompoovunoo-efvolof ooluifimunowouTunimmiemfmci000maf of ofifi Reafffompuoffiffuoleofomouomeiffofifioleofouipopimeiffofiefioomucompfaniviln 0g fioliolifaoalioiloofoviolioofoTeofogeofolia000lofoofoluiffaumofifolooliofoauflo fffiva off of filoge fuflofivialf 000moffilfofmou ffeoviof oauff of fififf fiof f ooffifiauf ovoiv ffiompfooffiunuffiffvolumfoofilofioofvfoffv000-efifoifoloTafaoffoa000fvogefo ffeuoioffeoofolifionfoof000fofoloffffeovofefufauffioiaTuffeovfoifiloiffoofuafia folouifouofaofaoluofovounfofeuomooaom000fioomoffoolailofouvofioffoffofvuofi gt u fioffIcovoovifeRefef oofloolofiloac oiov oiflooloiage of ff f oofifeuf of ffiviofioffioaf fe ufffoguaiouoifilfoaolofifiofuofofiloolifofffoaouooffioffifovioffofofeoffufaufRe ofi an flufl000fif fooifioaufooacuoifmolif f000f of ff goof ofuoifiof f oolifif oofoofvfloiofio ffovuougeou-couofffiaufmoffoivioffauffifffilofooffoololiffeofacofneffvfououaliai uofomfolafaTuffeaufefeuovfmoouofevw-coliofiumufl000nvuoufavolofoovifimuuo ot wouluniomileilifillepoomuffofofifm-effffoimouoffiffemeuomiofoomeTuRcommeufo foRemuunumemuflogefvuunuilffiviooffomaoofimaffemmufmoivioiffolovioomeo iouou-couafpueupplifilopuffigelumplifouomfuffilfoam000foimiffaufuefl000fovoofff ifeifouoliffiaifffeivfliouunuc000mfoloouoffaumofifemufoolifffem000lofffffommoi ofeuoif0000moffoofolifouoofolomoomooliomofomoolof000fofel000fogeoofilouomofoauf if g ofuof of omiffiffififf f off of ofeuiv of of f ofeifl000f of ouff fInf of flualoofeof ofilfuou-coo oil000foiaoauof000ffauaofumifoffiof000fom00000vocofeofiloofoTemiacu000mifoffioo anueff fioefifoifonommf oif oof fiouoiof of of ouivif oifaifeici000f oivu000mf ouff felooff uffmmoffafaifeifeufeoolviofefiolooffoloofooffefooffefeofiumummmulaufloffv000 ofoolow000foolif0000foolam0000f000v000foolom0000f000viofmoioffioifvomiovifInoi uoiouumoifmffifilfmoivofiouommeofeumunacomunouovofewuofunTuRcomiffiummiofe ofiTumfiimeffaufifaufeoviRefmauReofiloommuifeolviviiovoifvolifeifeifeDDyip IaLVVVVDDIDIDVaLDVILLIDDDVDVDIDDDIVDVDVIDVVIDDIDIDVDIDI
DilDiaLD333DIDIDIDy myypilofifefiloofilofeRevuoloofemiofioulooRefffeionio ffioiologefffioofeflovf000amiffioloioifffiouifilofimpfloiacuoefeufau-c000loumnioff gz fReffpuffffnueRefuemmiouoogeiplegeifiofuoffu-compufleupougumpouiffologefoileuf flopfooppippfoofffmooppleffoifufaegeopopfolippfolipifpfoolipipoffpfipioffoofpfl oofff Reffffoifilfiffif oomuTaiouofffiofiiffeloffffeauffiofiof000filoofloofoofovolocufeoffo uoof oTef 000l00000mof omouff flomoolocuoifioacoo-coofiv of f f fiof floe oppopumfoufpfilifif impfififfifoffifoReolfoolfilf000ffifilfuffalemopifiofilffioomuleifiloopopmifomo ffo 0z mf000liofivioficovifioloofmmofioficiafififiofommoolofilfmomioliciefloaivfnufif meRcupuileffipipanovulifipplifipipfpummifoopmmougumifiumeuaufReauppfileififfuol igui ouivuifimufffemmoumuovuoffffiffInu-eaucummifvuemuouvifffmuflimmifiolifilievo oommuoofoomiovogefoiTuageofiaufifeuouifiofefoeffiuoffoloiouovfffoofoofooalfolif e ffioflooiffvouovfofofeufame000aufnuogefl000foolf-coomogefloaciouomeoa000fiofiof g T
if0000ffoaofforc0000mmaeof000mouoaaoofologeofifogeoffoafaovanouoofoolacuo iimaif fouov of f ou-cfnfoofeuou fooffvommoif au-cocoa-0 ocuomou-couifef fiofeuacof f ffio ovoReoffou ffafRepipuf ov of f fualogef ov of omaiffl000uaaoff fufoiifuufif faoof of oo oefeuommeoffaufauffeuoiloilovoacofogeffeoolfacioffeuf000fvoofooignoliolioamofeo f Ravouomf000aciof000foliofifialooffouloffoliomooalfol000u000ffi000fif000fiofeuof fo 0 T
ouomofiovolifeufl000alofeuoff ouloouoofvf off fef off faof f ooifif ofeoiifuuouoof f ou-cuif aufoffoafiofaoiffloov000fiffiffffoo-colifiofeffaofffeuogefifaLy33y3oyyyDy3 DDOVVDVDVVDVDVDDDDIDDDVDVDDDVDDIDDDVIIaLLDIDDLLOVVDDDD
LLVaLDDIDDLLDDDLLDIDDLLLVDDLLLLLLDDIDLLDDaLDLLDDDLLLDLLDV
DDVDDID333DODOVVDDVDDDODVDDODVDDVDDVVIDDDVDIDOVV333000 g VOODDDIVOIDIVVDDLLODDVDDVDDIDVVaLDVDDVVIDIDDDIDDVVVVDD

aLDVDDDVDIDIDDDVDDVDDDVDVDDDVDDDIDDDIDIDOVVIDDIDIVDVDV
DVDDIDDDDVDDDVDVDDDLLDDIDVIDVDIVDDDODVDDDVDDODVDDVVVDV
9886SO/OZOZSI1LIDd L88960/1Z0Z OM

ctaccagcggtggatgatgccggatcaagagctaccaactattaccgaaggtaactggcttcagcagagcgcagatacc aaatact gacttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcct gttaccagtggc tgctgccagtggcgataagtcgtgtcttaccgggaggactcaagacgatagttaccggataaggcgcagcggtcgggct gaacggg gggacgtgcacacagcccagatggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgc cacgc ttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg ggga aacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcagggg ggcggagcctatg gaaaaacgccagcaacgcggccatttacggacctggccattgctggccattgctcacatgactacctgcgttatcccct gattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcg aggaagc ggaagagcgcccaatacgcaaaccgcctctccccgcgcgaggccgattcattaatgcagctggcacgacaggtacccga ctggaa agcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggattacactttatgatccggc tcgtatgag tgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctc actaaaggg aacaaaagctggagctgcaagcaggccattgcatacgagtatccatatcataatatgtacatttatattggctcatgtc caacattaccgc catgagacattgattattgactagttattaatagtaatcaattacggggtcattagacatagcccatatatgg SEO ID NO:19 CMV:
AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC
CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG
TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC
CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC
ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT
GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC
CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT
CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA
GGTCTATATAAGCAGAGCTCGTTTAGTGAACCG
SEO ID NO:20 5'R/U5:
GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT
GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG
ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC
SEO ID NO:21 PSI:
Tcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtga gtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagt attaagcgggggag SEQ ID NO:22 RRE:
Tccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggta caggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattga ggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacct SEQ ID NO:23 WPRE:
Cccatatttgttctgtttttcttgatttgggtatacatttaaatgttaataaaacaaaatgg tggggcaatcatttacatttttagggatatgtaattactagttcaggtgtattgccacaaga caaacatgttaagaaactttcccgttatttacgctctgttcctgttaatcaacctctggatt acaaaatttgtgaaagattgactgatattcttaactatgttgctccttttacgctgtgtgga tatgctgctttaatgcctctgtatcatgctattgcttcccgtacggctttcgttttctcctc cttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtccgtcaacgtg gcgtggtgtgctctgtgtttgctgacgcaacccccactggctggggcattgccaccacctgt caactcctttctgggactttcgctttccccctcccgatcgccacggcagaactcatcgccgc ctgccttgcccgctgctggacaggggctaggttgctgggcactgataattccgtggtgttgt cggggaagggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacg agtcggatctccctttgggccgcctccccgcctgga SEQ ID NO:24 3' PPT:
tttttaaaagaaaaggggggac SEQ ID NO:25 3' delta U3/R/U5 tggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctc tggttagaccagatctgagcctgggagctctctggctaactagggaacctactgcttaagcc tcaataaagcttgccttgagtgcttCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTA
ACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGC
SEQ ID NO:26 5V40 on:
Atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttt tatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggct tttttggaggcctagg SEQ ID NO:27 KANr:
Attgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggcta tgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcagg ggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgag gcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgt cactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcat ctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacg cttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtac tcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgc cagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacc catggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcga ctgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattg ctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctccc gattcgcagcgcatcgccttctatcgccttcttgacgagttcttctga SEQ ID NO:28 COLE1:
agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaa aaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaa ggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttag gccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacca gtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttacc ggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaa cgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaa gggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgaggga gcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttg agcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcg SEO ID NO:29 Full sequence of WAS Vec lentiviral vector transfer plasmid with codon optimized WASp open reading frame: (sequence is beginning from the CMV):

AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC
CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG
TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC
CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC
ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT
GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTC
CAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT
CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA
GGTCTATATAAGCAGAGCTCGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCAGATCTGAGC
CTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAG
TGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCC
TTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcccgaacagggacttgaaagcgaaaggg aaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcga ggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatg ggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggtta aggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctaga acgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggac agctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagca accctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagat agaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaat tgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagag cagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgca gcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaa caatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatca agcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctgggg atttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggag taataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaatta acaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaat gaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatag tttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcag acccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggaga gagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaactttta aaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaaca gacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgatcacga gactagcctcgagCAAACCTTGTTATCTGAGAGAAAGCTAGAGCTTCCTTCTTTCAGCCCCC
AGAGACAATGTGGCCAGGCTCCGGAGGGCTGGGAAGATGAGCAATGCTTGTGAGTCACCATG
ATAGGAAGCAGAAGGGTCAGGAAGTCCCTGGGAGCAAGGCTTAGGGTTAGGGTTAGGGAGAA
AACACTTGGGCCTGGAGGCTCGGGGCAGGCTTCCTAGAGGGGAGGGGTACTAAAAGCCACCA
AAACCAAGACAGCAATGAAAGTAACCTCTGGTCTTCCTAACTGCTCATTATACACTAATTAT
GATGCACTAGCATGCTAAGAGACACTTCCACCAGCGCCATGACAGTGCCATGGCAACATCAA
GAAGTCACCCCACATGGTCTAAAAAGGGGAAGAACCTTCAGTTCCCGGAATTGCCCACCCCT
TTCCTGGAAAACTCATGAATAATCCACCCCTTGTTTAGCATATAATGACCTGAAGGGTAAGG
GGGTGTGGAGGTTGTGAAGGCGGGAAGGGGGGTAGCCCCTTCACCAATGTAAACAAGGATGT
GGGTTCTGCGGCCACACTCTCCCCCGCCCTCCCCAGCGGCATTTCCAGCAAGTCACATGTCC
TGCGCACAGGCTGGGGGCCCCCTGCTGCTGCCTTTCTTAGAAGCCAGGACCACAGAGCCCGC
ACAGTGAGCTACTTGGGGAGCTATTTCTGTAGACTGAGCTTGGGGTACTGGGAGGCAGTGCT
GTTGGCGATCTGGGGGAAAACCCCCTGAGGGTCCCATTTCTCTGCCACTGACCCCTTTGCCT

GGCAGAAAGGC TCTGCT GC CTCC TAGC T GTGTAGCAATGGATAAGGC CTT TAACC TCTC TAA
GCT GAAAT GTC CT CAT GCATAAGGT GGAGACAGTAACTC CTCT TCCATCTGCT GGGT GI T GI
CAAAACTACACAGGAGGTT T GC CAAGCAC C TAAGG CACAGTAG GT GG T CAAGAAACAGGAAC
ACAATTTGTCATGACGACAATGATTCCTTCATATGCTAGTTACCCACGAGTCAGCCAGAATG
TTGCCATAACCACTTATGAAGCCCTTCCTCATTTCTGTTGCAATTCCTCAGCCAGAGGAAAG
CCC CACACAGC CCTT GCCACTCCCCCAT GGC CCAT GTAGAAACAAC CAT GI GGCC TTCACTC
T GC CCACAGAAGC TAGCAC CAGGACCC T GGT CAGGGT TAGAGGTTTC T GCT GAGT CAAAGCC
ACATGGAGGGAGGGAGCAAGGGAGAGATGCAGAGT CATGTTTC CAGGAGGAGGTTATCT GAG
CATAACAGGGACAGGGTGGGCCACAGGATACCTCTGAGGCTCAGGTTCCCACCTCCACTCCA
CCCTCCAACTCTCTGCTCCACGTTCCTTCCACTATTCAACACATGGCAGGAAGTCAGCCTGT
AGGGCTT CACAC TACAGTC TAAGCT GACT GC CATC T GTC CCCATCCAGGCGAGGCT GGAAAC
GGGGGCAGCAAAAAGCCCAGGGACAAAGTCC CCTT CCCAGGCACATGTAT TAT GCACTC T GC
ACCAAGGAAACCTCCAGATAAGGTCTAGAGACCAAGGGCCATGTACTTCGGGGGAGAGGCCA
GAGGACTTCTGAGGTTTTACAGAGAAAAGCCAAAGGCAGCCCAGTCAGGGGAAATGTGTAGC
CATAGTGCCGATAAGGAAAGGCCTTCAACCTGCCCTGGTCAGCTCTTCCTGTAAGTAGAGGC
CCCTTACCGAAGGCCCCAGTGGGAGGAAGGGTCGGGAGTActcgagTCAGCCTCAGGCTACC
TAGGT GCTT TAGAAAGGAGGCCACCCAGGCC CAT GAC TACTCC TT GC CACAGGGAGC CCT GC
ACACAGATGTGCTAAGCTCTCGCTGCCAGCCAGAGGGAGGAGGGTCTGAGCCAGTCAGAAGG
AGATGGGCCCCAGAGAGTAAGAAAGGGGGAGGAGGACCCAAGCTGATCCAAAAGGTGGGTCT
AAGCAGTCAAGTGGAGGAGGGTTCCAATCTGATGGCGGAGGGCCCAAGCTCAGCCTAACGAG
GAGGC CAGGCCCACCAAGGGGCC CCTGGAGGACTT GTTT CCCT T GTC CCTT GI GGTT TTTT G
CATTT CCTGTTCCCT T GCT GCTCATTGCGGAAGTT CCTC TTCT TACC CT GCAC CCAGAGCC T
CGCCAGAGAAGACAAGGGCAGAAAGCACCATGTCTGGCGGACCTATGGGAGGTAGACCTGGT
GGAAGAGGTGCCCCCGCCGTGCAGCAGAACATCCCCAGCACCCTGCTGCAGGACCACGAGAA
CCAGCGCCTGTTCGAGATGCTGGGCCGCAAGTGCCTGACCCTGGCCACCGCCGTGGTGCAGC
TGTACCTGGCCCTGCCCCCCGGCGCCGAGCACTGGACCAAGGAGCACTGCGGCGCCGTGTGC
T IC GI GAAGGACAACCCCCAGAAGAGC TACT TCAT CC GC CT GTACGGCCTGCAGGCC GGCC G
C CT GC T GTGGGAGCAGGAGCT GTACAGCCAGCT GGT GTACAGCACCCCCAC CC CCTT CTTC C
ACACCTTCGCCGGCGACGACTGCCAGGCCGGCCTGAACTTCGCCGACGAGGACGAGGCCCAG
GCCTTCCGCGCCCTGGTGCAGGAGAAGATCCAGAAGCGCAACCAGCGCCAGAGCGGCGACAG
AAGGCAACTGCCTCCTCCACCTACACCAGCCAACGAGGAAAGAAGAGGCGGACTGCCCCCCC
TGCCTCTTCATCCTGGCGGAGATCAAGGTGGACCTCCTGTGGGACCACTGTCTCTTGGCCTG
GCCACCGTGGACATCCAGAACCCCGACATCACCAGCAGCCGCTACCGCGGCCTGCCCGCCCC
CGGCCCCAGCCCCGCCGACAAGAAGCGCAGCGGCAAGAAGAAGATCAGCAAGGCCGACATCG
GCGCCCCCAGCGGCTTCAAGCACGTGAGCCACGTGGGCTGGGACCCCCAGAACGGCTTCGAC
GTGAACAACCTGGACCCCGACCTGCGCAGCCTGTTCAGCCGCGCCGGCATCAGCGAGGCCCA
GCTGACCGACGCCGAGACCAGCAAGCTGATCTACGACTTCATCGAGGACCAGGGCGGCCTGG
AGGCCGTGCGCCAGGAGATGCGCCGCCAGGAGCCCCTGCCACCACCTCCACCTCCATCTAGA
GGCGGCAACCAGCTGCCCCGCCCTCCTATCGTTGGCGGCAACAAGGGAAGATCTGGCCCTCT
GCCTCCTGTGCCTCTGGGAATCGCTCCACCACCACCAACACCTAGAGGCCCGCCTCCACCAG
GCAGAGGTGGTCCTCCGCCGCCACCTCCTCCAGCCACCGGCCGCAGCGGCCCCCTTCCTCCT
CCACCACCTGGTGCTGGTGGACCTCCAATGCCACCGCCACCGCCTCCGCCACCTCCGCCTCC
AAGTTCTGGAAATGGACCTGCTCCTCCTCCTTTGCCTCCTGCTTTGGTTCCTGCTGGCGGAT
TGGCTCCAGGCGGAGGAAGAGGCGCACTGCTGGACCAGATCCGCCAGGGCATCCAGCTGAAC
AAGACCCCTGGCGCTCCTGAGAGTTCTGCTCTGCAACCGCCACCACAGTCTAGCGAAGGACT
TGTGGGAGCCCTGATGCACGTGATGCAGAAGCGCAGCCGCGCCATCCACAGCAGCGACGAGG
GCGAGGACCAGGCCGGCGACGAGGACGAGGACGACGAGTGGGACGACTAActgcaggaatt c gagcatcttaccgccatttattcccatatttgttctgtttttcttgatttgggtatacattt aaatgttaataaaacaaaatggtggggcaatcatttacatttttagggatatgtaattacta gttcaggtgtattgccacaagacaaacatgttaagaaactttcccgttatttacgctctgtt cctgttaatcaacctctggattacaaaatttgtgaaagattgactgatattcttaactatgt tgctccttttacgctgtgtggatatgctgctttaatgcctctgtatcatgctattgcttccc gtacggctttcgttttctcctccttgtataaatcctggttgctgtctctttatgaggagttg fefogefigeolfefofeofofefooufoRefoofeofoofolofoomflogefifamoofoommfooemffifio iiefl0000mifofioomolifvocolofimooffiofmooffiooliffommooffofonofuoofouReuffmo ofeffoffffffeolfolofvfifiimefoifofailoufloioacoofoilifffoifioolfeimoviffloofanu fff og ffeooliofefffefouofofefeffemeffoifffeoffofeuiffooviffeaeffoffeRefefffeuf000mfou oofofnufaviofefifpfuompoulefufianfooupuipaufaucfpfuffilogeoppfuoupuofifolifffff f on fioff f oif f of-0 of of fnief foomifeief oefReopuf fliff foomioifif oifemuf of f ife oofiofiof f if000mifiooTemofiolofoloomomoofoouofeifioimefeuoliouoacooffmifeifoofeififeiolio lifiou ToRe oauefe of of-0 fe of-0 oliof fioumf fuefoomiopueompfufuuovff oofmf iiif fif f of-0 oomof oo gt uoounueReaucuofilofiofioTnif of ofioimmooiefefiloilovf fRouplegeReufuifoopaufuoifofuflo upplifomfaifoumpoom-eupaufleopfoolcifielunowouTumoimumfmm0000nf f of of ifTuu uf f ff oimou of fif foovofoouocomeiff ofifiov of amooloimuif fofvfloomeuomiof ocuivimfi oliolifef oefiloiloof ovioiloof ovof of-0 of olief 000lof oof oviff omofif oloomfoouflof ffvuf of f of filogefuuflofiviu fif 000mof flifofmouf fe oviof oauff of fifif f fiof f ooffifiauf ovolief f ot lomof ooffvueuf fiffvomn f oofilo floofiu f of f v000u fif oif oiov f fef off oef 000fiu of-0 f of f eeogabfieDaboggbgaeeboofyeDababogo .6.6.6.6eDgeofiefieeboe.6.6gogebgebfieogebogfyggog.6.6Dabee.6.6gebbogaegbaeo fyebabebogeobogeoeeebabeeDoeDoeboggeoDabgoaegobboogefyggaboegeo.6 qa6.63.6.63.6qeea6Tebqp.6.6qeoqepoqeqfieeefieboabqopqabqqopeoqoqeoqbqo g ogogebfieD.6.6.6.633.6gfiee.63.6.6.6ggegabga6.6goe.6.6fiee.6.6.6ofieebgoeogfygg boe bogabgbgabeababggoogq.63.6.6.6Debaeoa6.6go.6.6-4.6ogego.6.63.63.6eabfieboefye ea6goeebgeebg000fy4.6.633-4.6gooebooefieeogfyggqggogq.6.6333.63.6.6.6.6eabob eogbgabboogq.6-4.6DaboabgebgogabgabbogeeDefieDeeDea6.6.6goebgegabbog Teqabfiefie.6.6-4.6.6.6qqaboabbooqoqq.6.6eaboeofygqe.6.6q-efieepeefygqebTeofm N
ggbogebfiebgebfieoefiefieeogefiegoaeofiegeegeeoggabgeeegebgooDeegeeD
efiebgeogaboogegbgegeeeoggeoegeeegoggqggegggfygggeg000Dee.6.63.63.6g bgeee.6.6.6.6oggggoea6.6-4.6fiegggeeDeggaboeeggegeeeeDeeggggeebabapegg TeeeeeDeegggebgabebgeeeeeegg.6.6ggegoabbogggeboabgqgge.6.6fieegeggq efygqqqoqq-eqoq.6.63qoqeqoppeepqappeepee.6.6qapeepoqq.6qqoqoe.6.6-4.6eqee cz gggoggbaeoogfie.6.6-4-4.6Defygggooaboggqgq.6.6Defiegebgooabogeoa6.6.6gfieg.6 Deogq.6.6gefy4.6.6fieggebqqapeeeeeDooDebogoaeabboegggabgfiegggeboogg.6 .6fieggg000go.6.6.6.6.6ogeeegogabeeogbooDogggabboaboggbaeoabogogggoog g000ggogggabogggoogabooababegooababeDabggaeoegabooefy4.63.6eababo eqq.6.6-4.6.6-4.6-4.6.6.63.6.63.6ofieeqqeD.63.6.63.6eqbqopababoe.6.6.6qee.63.6.6qeebqop cc fieabofyggfyeDeeDooggooabogeboaeabooabfiefieebabegeeq.63.6.6gofieDabogg g000DogeoeofieabggoabogeeggoeeDoaegg.63.6.6gooDeeee.6.6.6goefy4.63-4.6Dee Degggq.63-4.633.6.6gaeogabababaeggegbogfyebgbegegooaboggeeDoaegbae.6.6 fiegoo.6.6e.6.6qqqqqqabbebfiefygfiegfieefieDoggegofiebgogoabbogoaboabfiebo abbefiea6Teqqq-eqqqqqqqqe-eqaebqa6.6qeoppabooqoqqeopabooqqfyeopaboo gT
gapegooDab000geoDaboogoeegooDab000gegabegoga6.6gogbgeogeggogegb geeDgeogoeeeDogfyggq.6.6-4.6ggfiegoggeabgaeoggqqqqqeofieeegeeeDeoggge eeDeogeofyegeeabeeeTeeeDegg.6.6geegeggabeofyggeggq.6qqapebfiefiefygfie6 efieogegeebgeeefyeeeofyggoeegegggegfieoggeggeggogeogbgeoggfyegfiegbe 3aV1313111,1VVaLaLV31V1111333VV313331VVI3VVI1313Vala= OT
,LaL3,1=3alalaLV,LITY3qq-06q-Bef)qq-006qq0Beeeqeepq-DoBeeqq-06q-Deq-00 ee.6.6fiegapega6.6gogogofie.6.6.6goofiebgogefieDoefiegg.6.6gogogog.6.6.6goegbq gabgqqqqabgogebeeDefieeboeeDoogoeoggeego.6.6fiee.6.6goe.6.6.6.6.6.6eeeefiee eeggqqqapoofieggogebegbgabeabfieeDeggoebgeeDoefieegggoaeg.6.6ogofieb oggee.6.6goabooDogoaboo.6.6.6ggg000goge.6.63-4.6eboefieogooaboggoaboggog g babooggogoo.6.63.6gogo.6.633.6gabgoo.6.6fiee.6.6.6.63-4.6qq.6-4.6.6-4.6Doggeegebgae 3.6.6.6gabgq.6.6ego.6.6.6.6eae.6.6gabgab000fyggoabgoaboabogeogoeefieabboeDo bogeb000gooDoogggabogggoe.6.6.6gogggoogapeogbqoaeoaeoofyggeo.6.6.6.6go .6.6goep000peeaboebgabggq.6-4.6gogabg.6-4.6.6-4.63.6.6-4.6apeogboogfyggbooD.6.6g 9886SO/OZOZSI1LIDd gaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgaggccgattcattaatgcagctggcacgacaggta cccgac tggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggcatacacatatgcac cggctcgt atgagtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaa ccctcactaa agggaacaaaagctggagctgcaagcaggccattgcatacgagtatccatatcataatatgtacatttatattggctca tgtccaacatt accgccatgttgac attgattattgactagttattaatagtaatc aattacggggtcattagttcatagcccatatatgg

Claims (71)

What is claimed is:

1. A recombinant lentiviral vector (LV) for the treatment of Wiskott-Aldrich Syndrome (WAS), said vector comprising:
an expression cassette comprising:
a nucleic acid encoding an effective fragment of the endogenous promoter of the WAS gene where said promoter has maximum length of 600 bp and contains the sequence of HS 1pro (SEQ ID NO:1); and a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp) operably linked to said effective fragment of the endogenous promoter of the WAS gene.

2. The vector of claim 1, wherein the sequence of said effective fragment of the endogenous promoter of the WAS gene consists of the sequence of HS 1pro (SEQ ID
NO:1).

3. The vector according to any one of claims 1-2, wherein said expression cassette comprises a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof.

4. The vector of claim 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID
NO:8).

5. The vector of claim 4, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).

6. The vector of claim 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).

7. The vector of claim 6, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment consists of the first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8).

8. The vector of claim 3, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the lst half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:3).

9. The vector according to any one of claims 3 and 8, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of the second half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4).

10. The vector according to any one of claims 3 and 8-9, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 2 of Enhancer Element 2 (SEQ ID
NO:5).

11. The vector according to any one of claims 3 and 8-10, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core Sub-Element 3 of Enhancer Element 2 (SEQ ID
NO:6).

12. The vector according to any one of claims 3 and 8-11, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 4 of Enhancer Element 2 (SEQ ID
NO:7).

13. The vector according to any one of claims 3 and 8-12, wherein said expression cassette comprises an effective fragment of enhancer element 2 wherein said fragment comprises or consists of Core-Sub Element 5 of Enhancer Element 2 (SEQ ID
NO:8).

14. The vector according to any one of claims 1-13, wherein said expression cassette comprises enhancer element H53 (SEQ ID NO:9) or an effective fragment thereof.

15. The vector of claim 14, wherein said expression cassette comprises an effective fragment of enhancer element HS3 wherein said fragment comprises or consists of HS3 core sequence (SEQ ID NO: 10).

16. The vector of claim 15, wherein said expression cassette comprises an effective fragment of enhancer element H53 wherein said fragment consists of H53 core sequence (SEQ ID NO: 10).

17. The vector according to any one of claims 1-16, wherein said expression cassette comprises enhancer element E9 (SEQ ID NO:11) or an effective fragment thereof.

18. The vector of claim 17, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment comprises or consists of enhancer element E9 core sequence (SEQ ID NO:12).

19. The vector of claim 18, wherein said expression cassette comprises an effective fragment of enhancer element E9 wherein said fragment consists of enhancer element E9 core sequence (SEQ ID NO:12).

20. The vector according to any one of claims 1-2, wherein said expression cassette comprises:
a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS 1pro (SEQ ID NO:1).

21. The vector of claim 20, wherein said vector comprises the features shown in Figure 18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

22. The vector of claim 20, wherein said vector comprises the sequence show in SEQ ID NO:15 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp)..

23. The vector according to any one of claims 1-2, wherein said expression cassette comprises:
enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO:12);

enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10);
a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS 1pro (SEQ ID NO:1).

24. The vector of claim 23, wherein said vector comprises the features shown in Figure 19 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

25. The vector of claim 23, wherein said vector comprises the sequence .. show in SEQ ID NO:16 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

26. The vector of claim 25, wherein said vector comprises the sequence show in SEQ ID NO:29.

27. The vector according to any one of claims 1-2, wherein said expression cassette comprises:I
enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO:12);
enhancer element H53 sequence comprising or consisting of H53 core sequence (SEQ ID NO: 10);
enhancer element 2 core sub-element 1 (SEQ ID NO:3 + SEQ ID
NO:4), enhancer element 2 core sub-element 4 (SEQ ID NO:7), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS 1pro (SEQ ID NO:1).

28. The vector of claim 27, wherein said vector comprises the features shown in Figure 20 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

29. The vector of claim 27, wherein said vector comprises the sequence show in SEQ ID NO:17 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

30. The vector according to any one of claims 1-2, wherein said expression cassette comprises:
enhancer element E9 sequence comprising or consisting of the E9 core sequence (SEQ ID NO:12);
enhancer element HS3 sequence comprising or consisting of HS3 core sequence (SEQ ID NO: 10);
a first half of enhancer element 2 core sub-element 1 (SEQ ID NO:3), and enhancer element 2 core sub-element 5 (SEQ ID NO:8); and a fragment of the endogenous promoter of the WAS gene consisting of the sequence of HS 1pro (SEQ ID NO:1).

31. The vector of claim 30, wherein said vector comprises the features shown in Figure 21 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

32. The vector of claim 30, wherein said vector comprises the sequence show in SEQ ID NO:18 where the sequence encoding mCitrine is replaced with a nucleic acid that encodes the Wiskott-Aldrich Syndrome protein (WASp).

33. The vector according to any one of claims 1-32, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS cDNA or a codon-optimized WAS gene.

34. The vector of claim 33, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a WAS cDNA (SEQ ID NO:13).

35. The vector of claim 33, wherein said nucleic acid that encodes a nucleic acid that encodes WASp protein is a codon optimized WAS.

36. The vector of claim 35, wherein the sequence of said nucleic acid that encodes WASP is a codon optimized WAS selected from the group consisting of jCAT codon optimized WAS, GeneArt optimized WAS, and IDT optimized WAS.

37. The vector according to any one of claims 1-36, wherein said vector comprises a iv region vector genome packaging signal.

38. The vector according to any one of claims 1-37, wherein said vector comprise a 5 LTR comprising a CMV enhancer/promoter.

39. The vector according to any one of claims 1-38, wherein said vector comprises a Rev Responsive Element (RRE).

40. The vector according to any one of claims 1-39, wherein said vector comprises a central polypurine tract.

41. The vector according to any one of claims 1-40, wherein said vector comprises a post-translational regulatory element.

42. The vector of claim 41, wherein the posttranscriptional regulatory element is modified Woodchuck Post-transcriptional Regulatory Element (WPRE).

43. The vector according to any one of claims 1-42, wherein said vector is incapable of reconstituting a wild-type lentivirus through recombination.

44. The vector according to any one of claims 1-43, wherein said vector shows high expression in megakaryocytes.

45. The vector according to any one of claims 1-44, wherein said vector restores T, B and NK cell counts and function when administered to a mammal having WAS.

46. A host cell transduced with a vector according to any one of claims 1-45.

47. The host cell of claim 46, wherein the cell is a stem cell.

48. The host cell of claim 47, wherein said cell is a stem cell derived from bone marrow, and/or from umbilical cord blood, and/or from peripheral blood.

49. The host cell of claim 46, wherein the cell is a human hematopoietic progenitor cell.

50. The host cell of claim 49, wherein the human hematopoietic progenitor cell is a CD34+ cell.

51. A method of treating Wiskott-Aldrich Syndrome (WAS), in a subject, said method comprising:
transducing a stem cell and/or progenitor cell from said subject with a vector according to any one of claims 1-45; and transplanting said transduced cell or cells derived therefrom into said subject where said cells or derivatives therefrom express said WASp protein.

52. The method of claim 51, wherein the cell is a stem cell.

53. The host cell of claim 51, wherein said cell is a stem cell derived from bone marrow.

54. The method of claim 51, wherein the cell is a human hematopoietic stem and progenitor cell.

55. The method of claim 54, wherein the human hematopoietic progenitor cell is a CD34+ cell.

56. A recombinant nucleic acid comprising one or more of the following:
a nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HS 1pro (SEQ ID NO:1); and/or a nucleic acid sequence comprising or consisting of a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof;
a nucleic acid sequence comprising or consisting of a 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO: 3) ; and/or a nucleic acid sequence comprising or consisting of a 2nd half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4); and/or a nucleic acid sequence comprising or consisting of a Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5); and/or a nucleic acid sequence comprising or consisting of a Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6); and/or a nucleic acid sequence comprising or consisting of a Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7); and/or a nucleic acid sequence comprising or consisting of a Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8); and/or a nucleic acid sequence comprising or consisting of enhancer element H53 (full) (SEQ ID NO:9) or an effective fragment thereof; and/or a nucleic acid sequence comprising or consisting of Enhancer element H53 core (SEQ ID NO:10); and/or a nucleic acid sequence comprising or consisting of Enhancer element E9 (full) (SEQ ID NO:11) or an effective fragment thereof; and/or a nucleic acid sequence comprising or consisting of Enhancer element E9 core (SEQ ID NO:12).

57. The nucleic acid of claim 56, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a minimal endogenous promoter of the WAS gene said minimal endogenous promoter comprising or consisting of HS 1pro (SEQ ID
NO:1).

58. The nucleic acid of claim 56, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a slim enhancer element 2 (SEQ ID NO:2 = SEQ ID NOs:3-8) or an effective fragment thereof.

59. The nucleic acid of claim 56, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 1st half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO: 3).

60. The nucleic acid according to any one of claims 56, and 59, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a 2nd half of Core Sub-Element 1 of Enhancer Element 2 (SEQ ID NO:4).

61. The nucleic acid according to any one of claims 56, and 59-60, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 2 of Enhancer Element 2 (SEQ ID NO:5).

62. The nucleic acid according to any one of claims 56, and 59-61, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core Sub-Element 3 of Enhancer Element 2 (SEQ ID NO:6).

63. The nucleic acid according to any one of claims 56, and 59-62, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 4 of Enhancer Element 2 (SEQ ID NO:7).

64. The nucleic acid according to any one of claims 56, and 59-63, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of a Core-Sub Element 5 of Enhancer Element 2 (SEQ ID NO: 8).

65. The nucleic acid according to any one of claims 56, and 59-64, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of enhancer element HS3 (full) (SEQ ID NO:9) or an effective fragment thereof.

66. The nucleic acid according to any one of claims 56, and 59-65 , wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element HS3 core (SEQ ID NO:10).

67. The nucleic acid according to any one of claims 56, and 59-66, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 (full) (SEQ ID NO:11) or an effective fragment thereof.

68. The nucleic acid according to any one of claims 56, and 59-67, wherein said nucleic acid comprises a nucleic acid sequence comprising or consisting of Enhancer element E9 core (SEQ ID NO:12).

69. The nucleic acid according to any one of claims 56-68, wherein said nucleic acid comprises an expression cassette.

70. The nucleic acid of claim 69, wherein said expression cassette is effective to express WASp when transduced into a mammalian cell.

71. The nucleic acid of claim 56, wherein said nucleic acid comprises a vector according to any one of claims 1-45.